DE69331957T2 - COMPOSITIONS CONTAINING ANTI-INFLAMMATORY FACTOR, METHOD FOR PRODUCTION AND USE - Google Patents

Abstract

The invention relates to an anti-inflammatory factor isolated from milk and to methods for using this factor to remove adhered neutrophils from endothelial cells, to prevent the emigration of cells from the vasculature and to suppress the response of lymphocytes to foreign antigens.

Description

BACKGROUND OF THE INVENTION Field of the invention

The present invention relates to an anti-inflammatory factor, a process for its preparation in substantially pure form, and a method for use in the treatment of inflammation.

Description of the related field

Inflammation, as defined in Dorland's Medical Dictionary, is "a local protective response elicited by injury or destruction of tissue which serves to destroy, dilute or isolate both the injuring agent and the injured tissue." It is characterized by fenestration of the microvasculature, leakage of blood elements into the interstitial spaces, and migration of leukocytes into the inflamed tissue. At a macroscopic level, this is usually accompanied by the familiar clinical signs of flushing, edema, hyperalgesia, and pain. During this complex response, chemical mediators such as histamine, 5-hydroxytryptamine, various chemotactic factors, bradykinin, leukotrienes, and prostaglandins are released locally. Phagocytic cells migrate to the area, and lysosome cell membranes may rupture, releasing lytic enzymes. All of these events can contribute to the inflammatory response.

Inflammation in patients with rheumatoid arthritis probably involves the combination of an antigen (gammaglobulin) with an antibody (rheumatoid factor) and complement, causing the local release of chemotactic factors, which attracts leukocytes. The leukocytes phagocytose the antigen-antibody complexes and also release the many enzymes contained in their lysosomes. These lysozymes then cause injury to the cartilage and other tissues, and this increases the extent of inflammation. Cellular immune responses may also be involved. Prostaglandins are also released during this process.

Prostaglandins, which are readily released during inflammation, cause skin flushing and increase local blood flow. Two important vascular effects of prostaglandins are not generally shared by other mediators of inflammation, a long-lasting vasodilator effect and a property of counteracting the vasoconstrictor effects of substances such as norepinephrine and angiotensin.

A number of mediators of inflammation increase vascular permeability (leakage) in the postcapillary and collecting venules. In addition, the migration of leukocytes into an inflamed area is an important aspect of the inflammatory process.

The Arthus reaction is an inflammatory response caused by the formation of immune complexes at subcutaneous sites where an antigen complexes with an antibody to that antigen. Neutrophil leukocytes characteristically bind to the Fc portion of the immunoglobulin complex that forms the subcutaneous injection site, where they release digestive enzymes that initiate the visible acute inflammation. Thus, the response is primarily neutrophilic and agents that induce the development of the response therefore act on these cells.

There are several ways in which an agent could impair neutrophil migration from blood vessels to a site of inflammation. One likely way is through inhibition of the leukocyte wall, the reversible "adhesion" of inflamed cells to the endothelial cell lining of the blood vessel wall. In the normal state, approximately 50% of neutrophils are reversibly adherent, but during an acute inflammatory response, adhesion becomes much stronger and is a key step in the process of neutrophil migration. While prostaglandins are unlikely to be directly involved in the chemotactic response, another product of arachidonic acid metabolism, leukotriene, is a very potent chemotactic agent.

The anti-inflammatory response is any response characterized by inflammation as defined above. It is well known to those of ordinary skill in the medical field that the inflammatory response causes much of the physical discomfort, i.e., pain and loss of function, that accompanies various diseases and injuries. Accordingly, it is common medical practice to administer pharmacological agents that have the effect of neutralizing the inflammatory response. Agents that have this property are classified as anti-inflammatory drugs. Anti-inflammatory drugs are used to treat a wide range of diseases and the same drugs are often used to treat other diseases. Treatment with anti-inflammatory drugs is not directed at the disease, but mostly at the symptoms, ie the inflammation.

The anti-inflammatory, analgesic and antipyretic drugs are a heterogeneous group of compounds that are often chemically unrelated but nevertheless share certain therapeutic effects and side effects. Corticosteroids represent the most commonly used class of compounds for the treatment of the anti-inflammatory response. Proteolytic enzymes represent another class of compounds thought to have anti-inflammatory effects. Hormones that directly or indirectly cause the adrenal cortex to produce and secrete steroids represent another class of anti-inflammatory compounds. A number of non-hormonal anti-inflammatory agents have been described. Among these, the salicylates are the most commonly used. Acetylsalicylic acid or aspirin is the most commonly described analgesic-antipyretic and anti-inflammatory agent. Examples of steroidal and nonsteroidal anti-inflammatory agents are listed in Physician's Desk Reference, 1987 (see pages 207 and 208 for a list of such preparations).

Natural and synthetic corticosteroid preparations cause a number of serious side effects, including increased blood pressure, salt and water retention, and increased excretion of potassium and calcium. In addition, corticosteroids can mask the signs of infection and increase the spread of infectious microorganisms. These hormones are approved for use in pregnant women. Women are not considered safe, and long-term corticosteroid treatment is associated with gastric hyperactivity and/or peptic ulcers. Treatment with these compounds may also aggravate diabetes mellitus, requiring higher doses of insulin, and may induce psychotic disorders. Hormonal anti-inflammatory agents that indirectly increase the production of endogenous corticosteroids have the same potential for adverse side effects.

Nonhormonal anti-inflammatory agents are synthetic biochemical compounds that can be toxic at high doses, with a wide range of undesirable side effects. For example, salicylates contribute to the serious disturbances of acid-base balance that characterize the toxic effects of this class of compounds. Salicylates stimulate respiration directly and indirectly. Toxic doses of salicylates cause central respiratory paralysis as well as circulatory collapse secondary to vasomotor depression. Salicylate ingestion can cause epigastric pain, nausea, and vomiting. Salicylate-induced gastric bleeding is well known. Salicylates can cause liver damage and prolong clotting time. Therefore, aspirin should be avoided in patients with severe liver damage, hypoprothrombinemia, vitamin K deficiency, or hemophilia, because inhibition of platelet hemostasis by salicylates can lead to bleeding. Salicylate intoxication is well known, and over 10,000 cases of serious salicylate intoxication are identified each year in the United States, some of which are fatal, and many of which occur in children. See Goodman Gilman's "The Pharmacological Basis of Therapeutics," 7th ed., 1985. Accordingly, despite the variety of anti-inflammatory agents currently available, there is still a need for a safe, effective anti-inflammatory product that is free from side effects and adverse reactions.

If a natural food product, such as one derived from milk, could be obtained with anti-inflammatory effects, this would be an easily administered, readily available and safe therapeutic composition.

It is known in the art to produce milk with a variety of therapeutic effects. For example, Beck has disclosed a milk containing antibodies against Streptococcus mutans which has a dental caries inhibiting effect (US Patent No. 4,324,782). The milk is obtained by immunizing a cow with S. mutans antigen in two steps and obtaining therapeutic milk therefrom.

Stolle et al. disclosed a method for treating smoking-related vascular disorders or pulmonary disorders in an animal which comprises administering to the animal animal milk collected from a cow maintained in a hyperimmune state (US Patent No. 4,636,384). Beck has disclosed a method for treating inflammation in an animal which comprises administering to the animal an anti-inflammatory effective amount of milk collected from a cow maintained in a state of producing an anti-inflammatory factor (US Patent No. 4,284,623). Heinbach, US Patent No. 3,128,230, has described milk containing globulins of alpha, beta and gamma components by inoculating a cow with antigen mixtures. Peterson et al. (US Patent No. 3,376,198), Holm (US application (published no. 628,987), Tunnah et al. (British Patent No. 1,211,876) and Biokema SA (British Patent 1,442,283) have also described antibody-containing milk.

However, none of the above references disclose the identity of the component or components of the therapeutic milk that produces the desired therapeutic effect. For example, the milk products used as a therapeutic agent in Beck, U.S. Patent No. 4,284,623 consist of either liquid whole milk, liquid nonfat whey, or whole milk powders. Although each of these milk products has anti-inflammatory properties, the factor or factors that actually provide the therapeutic benefit have not yet been isolated or identified.

Summary of the invention

The present invention relates to an anti-inflammatory factor contained in milk and to various methods involving the use of the anti-inflammatory factor contained in milk. In general, the invention relates to a composition comprising an anti-inflammatory factor, wherein the anti-inflammatory factor is approximately at least 55,000 times more purified than the factor in a 10,000 dalton skim milk filtrate tested in the mouse neutrophil migration inhibition assay, the composition being prepared by a method comprising:

(a) removing fat from the milk of a milk-producing animal to produce a skimmed milk solution;

b) filtering the skim milk solution from step a) through a filter to produce a skim milk filtrate having a molecular weight < 10,000, the filter retaining molecules having molecular weights greater than about 10,000 Daltons;

c) fractionating the skimmed milk filtrate produced in step b by ion exchange chromatography, so as to produce ion exchange fractions enriched in the factor;

d) purifying the factor from the enriched ion exchange fractions by gel filtration chromatography to produce gel filtration fractions further enriched in the factor;

e) purifying the factor from the enriched gel filtration fractions by affinity chromatography to produce a composition containing the anti-inflammatory factor that is 55,000-fold more purified than the factor in the skim milk filtrate from step b, tested in the mouse neutrophil migration inhibition assay.

The invention further relates to a composition for detaching neutrophils which have attached to endothelial cells in a mammal; a composition for preventing the interaction between CD18 cell surface antigens present in a mammal and other molecules; a composition for preventing the emigration of cells of the venous system; and a composition for suppressing the mitogenic response of lymphocytes in a host mammal to foreign antigens, which compositions comprise the above composition as an active ingredient, and which is prepared by a process comprising:

(i) removing fat from milk of a milk-producing animal to produce skimmed milk;

(ii) pasteurizing the skimmed milk;

(iii) removing casein from the pasteurised skim milk to produce whey;

(iv) removing macromolecules having a molecular weight greater than about 10,000 daltons from the whey to produce a composition free of macromolecules having a molecular weight greater than about 10,000 daltons;

(v) reducing the ionic strength of the composition of step (iv) to produce an aggregate having anti-inflammatory activity, wherein the aggregate has a molecular weight of greater than about 5000 daltons;

(vi) removing macromolecules having a molecular weight of less than about 5,000 daltons from the composition of step (v) to produce a composition free of macromolecules having a molecular weight of less than about 5,000 daltons;

(vii) collecting the composition from step (vi).

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendant advantages will be readily obtained by reference to the following detailed description in conjunction with the accompanying drawings, where:

Fig. 1. Isolation of the anti-inflammatory factor by ion exchange chromatography on a column of DEAE-cellulose.

Fig. 2. Fractionation of the anti-inflammatory factor-containing peak (second) from DEAE-cellulose chromatography (Fig. 1) on a Sephadex G-10 molecular sieve column.

Fig. 3. Effect of immune milk on carrageenan-induced edema in rats (paw weight, % control paw, mean ± sem, n = 10).

Fig. 4. Effect of intraperitoneal administration of anti-inflammatory factor on paw edema in rats (ul, mean ± SD, n = 6).

Fig. 5. Response curve to an intraperitoneal dose of anti-inflammatory factor in the rat paw edema test (% control, mean ± SD, n = 6).

Fig. 6. Effect of hyperimm-milk factor versus placebo (lactose) on paw edema in rats (% control, mean ± SD, n = 6).

Fig. 7. Effect of iv and oral MAIF on paw edema in rats (% control, mean ± SD, n = 6).

Fig. 8. Effect of low iv dose of MAIF on paw edema in rats (% control, mean ± SD, n = 6).

Fig. 9. Intravenous dose response curve for MAIF in the rat paw edema test (PS control, mean ± SD, n = 6).

Fig. 10. Run 1, Double-foci/ultrafiltration experiments (% mean control edema, mean ± SD, n = 6).

Fig. 11. Run 2, Double-foci/ultrafiltration experiments (% mean control edema, mean ± SD, n = 6).

Fig. 12. Run 3, Double-foci/ultrafiltration experiments (% mean control edema, mean ± SD, n = 6).

Fig. 13. Effect of different treatments of MAIF on inhibition of paw edema in rats (ul paw edema, mean ± SD, n = 6).

Fig. 14. Effect of fractions of MAIF and of immune-wpc on inhibition of paw edema in rats (ul paw edema, mean ± SD, n = 6).

Fig. 15. Effect of five different anesthetics on the response to carrageenan in the rat paw. Edema accumulation was monitored at selected intervals in the same animals. n = 6 for each data point.

Fig. 16. Demonstration of the biphasic nature of the response to carrageenan in the rat paw. n = 5 for each data point. Ether was used as the anesthetic.

Fig. 17. MAIF administered at either 5 mg per rat (A) or 40 mg per rat (B) does not inhibit the inflammatory response to carrageenan in ether-anesthetized rats. n = 4 for all data points.

Fig. 18. Suppression of carrageenan-induced edema accumulation during the secondary, phagocytic cell-mediated response by 40 mg of MAIF injected i.v. at the time of carrageenan challenge (time 0). n = 12 for each data point in the control group and n = 10 for each data point in the MAIF-treated group.

Fig. 19. Effect of MAIF, administered i.v. at 4 mg per rat at various times, on the response to carrageenan in the rat paw. Edema was assessed in all cases 4 hours after challenge. n = 12 for each data point.

Fig. 20. Effect of 20 mg of MAIF injected IV on the reverse passive Arthus response. * = p < 0.01; ** p < 0.05.

Fig. 21. Effect of decreasing doses of MAIF on the ability of neutrophils to migrate from the vasculature into subcutaneously implanted sterile sponges. * = p < 0.01.

Fig. 22. Effect of MAIF administered at a dose of 20 mg per rat to inhibit the ability of inflammatory cells to accumulate in subcutaneously implanted sponges when administered at the time of implantation or up to 120 minutes after implantation. * = p < 0.01.

Fig. 23. Time course of infiltration of inflammatory cells into subcutaneously implanted sponges in normal animals.

Fig. 24. Effect of anti-inflammatory factor preparations on platelet activating factor (PAF) induced adhesion of neutrophils to venules.

Fig. 25. Effect of anti-inflammatory factor preparations on PAF-induced neutrophil emigration.

Fig. 26. Effect of preparations of anti-inflammatory factor on PAF-induced flow of neutrophils through venules.

Fig. 27. Reversal of neutrophil adhesion by preparations of anti-inflammatory factor. Figure 27a shows the effect of the MAIF preparation (40 mg/rat) in reducing the number of neutrophils adhering to venules in response to PAF. Figure 27b shows the effect of the MAIF preparation (40 mg/rat) on new neutrophil-endothelial cell adhesions.

Fig. 28. Effect of anti-inflammatory factor preparations on the velocity of neutrophils in venules.

Fig. 29. Effect of anti-inflammatory factor preparations on the velocity of red blood cells in venules.

Fig. 30. Effect of anti-inflammatory factor on leukocyte flow in venules.

Fig. 31. Effect of 40 mg of the MAIF preparation administered i.v. on the number of circulating neutrophils and lymphocytes in the 24 hours following the injection.

Fig. 32. Dose-response relationship between i.v. administration of MAIF preparation and the number of circulating leukocytes (p < 0.01).

Fig. 33. Effect of anti-inflammatory factor on various aspects of lymphocyte function. Figure 33a shows the effect of prior administration of the factor on the response of host T lymphocytes to foreign histocompatibility antigens. Figure 33b shows the results obtained when lymphocytes from MAIF-treated rats are injected into untreated rats. Figures 33C and 33D show the effect of MAIF treatment on spleen weight and spleen cell number in rats. Figure 33E shows the effect of MAIF treatment on the concanavalin A stimulated mitogen response of lymphocytes.

Fig. 34. Suppression of infection-induced edema by 40 mg of MAIF injected i.v. The means of the two groups were: control, 87 ± 22 µl; MAIF, 45 ± 17 µl; p < 0.01.

Fig. 35. Effect of IV MAIF at 40 mg per rat on bacterial replication and subcutaneously implanted E. coli-infected sponges.

Fig. 36. Inhibition of infiltration of inflammatory cells into infected sponges by MAIF (40 mg per rat, i.v.).

Fig. 37. Effect of MAIF (40 mg per rat, i.v.) on suppression of the interphase (4-16 hours) of inflammatory fluid accumulation in E. coli-infected sponges.

Fig. 38. Effect of 40 mg of MAIF given intravenously at the time of irritation and 48 hours later on the pathogenesis of experimental pyelonephritis. The dotted line on the left graph represents the mean background kidney weight. * = p <0.01; ** = p < 0.02.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention encompasses the isolation and purification of an anti-inflammatory factor from milk and the administration of the factor to an animal for the purpose of treating anti-inflammatory disorders. Unless otherwise stated, the following definitions are used:

The term "milk anti-inflammatory factor" refers to a factor obtained from either hyperimmune milk or normal cow's milk. The term "substantially pure milk anti-inflammatory factor" within this invention refers to an anti-inflammatory factor that, after removal of the high molecular weight substances (> 10,000 daltons) and isolation of the negatively charged low molecular weight species by ion exchange chromatography, elutes as a single symmetrical major peak in HPLC chromatography. Both normal milk and hyperimmune milk can be processed by the methods described herein to obtain the anti-inflammatory factor.

The term "hyperimmune milk" refers within this invention to milk from milk-producing animals that are maintained in a hyperimmune state, the details of hyperimmunization being described in more detail later.

The term "whey" in this invention refers to milk from which the cream has been removed.

The term "normal milk" refers within this invention to milk obtained from milk-producing animals by conventional means and dairy processes.

The term "milk-producing animal" refers within this invention to mammals that produce milk in commercially possible quantities, preferably cows, sheep and goats, more preferably dairy cows of the genus Bos (bovid), especially those breeds that give the highest milk yields, such as Holstein.

The term "bacterial antigen" refers within this invention to a lyophilized preparation of heat-killed bacterial cells.

The term "microencapsulated form" refers within this invention to polymeric microparticles encapsulating one or more bacterial antigens for administration to dairy animals.

The term "inflammation" within this invention refers to a local protective response triggered by injury or destruction of tissues, which serves to destroy, dilute or isolate both the injurious agent and the damaged tissue, which in the acute form is characterized by the classic sequence of pain, warmth, redness, swelling and loss of function and histologically involves a complex series of events, including dilation of arterioles, capillaries and venules with increased permeability and blood flow, exudation of fluids including plasma proteins and leukocyte migration into the core area of inflammation.

The term "treating" within this invention refers to the improvement or complete elimination of the signs of the disorder and/or the pathogenic origin of the disorder.

The term "administering" within this invention refers to any method of treating a living being with a substance, such as orally, intranasally, parenterally (intravenously, intramuscularly or subcutaneously) or rectally.

The term "animal" in this invention refers to any living being that is subject to inflammation, including humans, livestock, pets or zoo animals.

Examples of inflammatory conditions that can be treated by the isolated and purified milk product of the present invention are conditions selected from the group consisting of acute and subacute bursitis, acute nonspecific tendinitis, systemic lupus erythematosus, systemic dermatomyositis, acute rheumatic carditis, pemphigus, bullous dermatitis, herpeteformis, severe erythema, multiform exfoliative dermatitis, cirrhosis, intermittent perennial rhinitis, bronchial asthma, ectopic dermatitis, serum sickness, keratitis, ophthalmic iritis, diffuse ureitis, chorditis, optic neuritis, ophthalmia sympatica, symptomatic sarcoidosis, Loeffler syndrome, berylliosis, hemolytic anemia, mastitis, mastoiditis, contact dermatitis, allergic conjunctivitis, psoriatic arthritis, spondylarthritis, acute gouty arthritis and herpes zoster. Furthermore, the isolated and purified milk product can be used to treat animals exposed to potential inflammatory agents.

The invention is based in part on the discovery that when a milk-producing animal such as a bovid is placed in a specific state of hyperimmunization, the animal will produce milk containing supranormal levels of the very useful anti-inflammatory factor, which factor not only suppresses the symptoms of inflammation in humans and other animals, but also is a prophylactic agent against the presence of inflammatory agents in the recipient. The term "supranormal levels" refers to levels above those found in milk from non-hyperimmunized animals. Induction of immune sensitivity alone is insufficient to cause the appearance of supranormal levels of MAIF in milk, as demonstrated by the fact that normal cows' milk does not contain these supranormal levels, even when cows have been sensitized to various antigens during normal immunization against cow diseases and during normal environmental exposure. It is only in specific hyperimmune states that milk contains the desired supranormal levels.

This particular condition can be achieved by administering an initial immunization followed by periodic boosters with sufficiently high doses of specific antigens. The preferred dosage of booster should be equal to or greater than 50% of the dosage required for primary immunization of the bovid. Thus, there is a limit of booster dose below which the traits will not be produced in the milk, even if the cow is in a state which is usually referred to as an immune state. To achieve the required hyperimmune state, it is essential to test the hyperimmune milk after an initial series of booster administrations. If the beneficial factors are not present in the milk, additional boosters of high dosage are administered until the property appears in the milk.

The process of producing hyperimmune milk containing supranormal levels of an anti-inflammatory factor is disclosed in US Patent 5,106,618 and US Patent 4,919,929.

In summary, a method of producing hyperimmune milk containing supranormal levels of an anti-inflammatory factor comprises the following steps:

(1) Antigen selection;

(2) primary immunization of the bovid;

(3) Testing of serum to confirm sensitivity induction;

(4) Hyperimmunization with boosters of appropriate dosage; and optionally

(5) Testing milk for anti-inflammatory properties;

(6) collecting milk from the hyperimmune bovid; and

(7) Processing the milk to isolate the MAIF.

Step 1:

Any antigen or combination of antigens can be used. The antigens can be bacterial, viral, protozoal, fungal, cellular or any other substance to which the immune system of a dairy animal will react. The critical point in The key to this step is that the antigen(s) must be capable of not only inducing immune and hyperimmune states in the milk-producing animal, but also of producing supranormal levels of anti-inflammatory factor in the milk. Any antigen may be used to produce supranormal levels of a factor. A preferred vaccine is a mixture of polyvalent bacterial antigens, referred to as the 100 series vaccine, which is described in detail in Example 1A below.

Step 2:

The antigen(s) may be administered by any method that causes sensitization. In one method, a vaccine composed of an antigen derived from 1 x 106 to 1 x 1020, preferably 108 to 1010, most preferably 2 x 108, of heat-killed bacteria is administered by intramuscular injection. However, other methods such as intravenous injection, intraperitoneal injection, rectal suppository or oral administration may be used.

Step 3:

It is necessary to determine whether or not the milk-producing animal has become sensitive to the antigen. There are a number of methods known to those skilled in the art of immunology to test for sensitization (Methods in Immunology and Immunochemistry, William, CA and Chase, WM, Academic Press, New York, Volumes 1-5 (1975)). The preferred method is to use a polyvalent vaccine comprising multiple bacterial species as the antigen and to test for the presence of agglutinating antibodies in the serum of the animal before and after challenge with the vaccine. The appearance of milk antibodies after Immunization with the vaccine indicates sensitization; at this point it is possible to proceed to step 4.

Step 4:

This involves the induction and maintenance of the hyperimmune state in the sensitized animal. This is accomplished by repeated booster administration of the same polyvalent vaccine used to achieve primary sensitization at specified intervals. A booster interval of two weeks is optimal for polyvalent bacterial antigens. However, it is necessary to ensure that the animal does not move from a hyperimmune state to a state of immune tolerance to the antigen.

In a preferred embodiment, hyperimmunization of bovids can be achieved by a single administration of microencapsulated vaccine prepared as detailed in Example 1B below. The advantage of the controlled release form of hyperimmunization is that the constant exposure to antigen ensures that the animal remains in the hyperimmune state.

In an alternative embodiment, it is also possible to combine different immunization methods, e.g. simultaneous administration of microencapsulated and liquid antigen, or intramuscular injection for primary immunization and booster doses by oral administration or parenteral administration of microencapsulating agent. Many other combinations of primary immunization and hyperimmunization are known to those of ordinary skill in the art.

Step 5:

It is necessary to test the milk for anti-inflammatory activity levels. This can be accomplished by any assay that tests the effects of either the hyperimmune milk or products derived from it on inflammation. Chemically induced inflammation of the rat's foot is a standard test for anti-inflammatory drugs.

Step 6:

This involves collecting and processing the milk. The milk can be collected using conventional methods. Processing the milk to isolate the anti-inflammatory factor is described below.

The simplest procedure for isolating, purifying and testing the anti-inflammatory factor involves the following steps:

1. Defatting the hyperimmune milk to produce skim milk;

2. Removing casein from skim milk to produce whey;

3. Removal of macromolecules with a molecular weight greater than approximately 10,000 Daltons by ultrafiltration from the whey;

4. Fractionating the product of step 3 using an ion exchange resin column to isolate a negatively charged anti-inflammatory species having a molecular weight of less than about 10,000 daltons;

5. Separating the negatively charged species from step 4 by molecular sieve chromatography; and

6. biological assay of the preparation of the anti-inflammatory factor from step S.

In an alternative preferred embodiment, the fractions of molecular sieve chromatography that possess biological activity are further purified by filtration through a membrane that retains macromolecules having a molecular weight greater than about 5000 Daltons.

7. The anti-inflammatory effect of milk factor is tested on edema caused by an injection of a solution of carrageenan into the paw of rats. The rat paw test is the standard animal test for anti-inflammatory drugs. Winter, CA, Risley, GA, Nuss, AW, "Carrageenan-Induced Edema in the Hind Paw of the Rat as an Assay for Anti-inflammatory Drugs" Proc. Soc. Exper. Biol. Med. 3: 544 (1967). A variety of other tests may be used. Wetnick, AS, and Sabin, C., "The Effects of Clonixin and Bethaurethasone on Adjuvant-Induced Arthritis and Experimental Allergic Encephalomyelitis in Rats" Jap. J. Pharm. 22: 741 (1972). However, the rat paw test is the simplest and most direct test available and has been shown to be satisfactory for all anti-inflammatory drugs. This test is described in detail in Beck, U.S. Patent 4,284,623, which is incorporated herein by reference insofar as it describes the rat paw test. Briefly, the test involves injecting a small amount of carrageenan into the paw of adult white rats. This is known to induce an inflammatory response. The resulting degree of swelling can be quantified. Samples containing an anti-inflammatory factor are administered to the rat by an appropriate route, preferably by intraperitoneal injection, and the blockade or amelioration of the inflammatory process quantified by either volumetric or gravimetric methods.

In summary, one can thus isolate the anti-inflammatory factor from hyperimmunized milk by following a procedure of defatting the milk, removing the casein, removing the macromolecules larger than 10,000 daltons, followed by ion exchange and molecular sieve chromatography. The biological activity of suitable preparations of an anti-inflammatory factor can be tested by conducting a dose response experiment in rats as described herein.

In an additional preferred embodiment of the present invention, the anti-inflammatory factor present in hyperimmunized milk is purified using a combination of steps comprising: filtration on a membrane capable of separating molecules based on their molecular weight; ion exchange chromatography; molecular sieve chromatography; and affinity chromatography (Example 15).

The preferred first step involves filtering hyperimmune skim milk, prepared as described above, through a membrane which retains molecules with molecular weights of approximately 10,000 daltons or more. The material passing through the membrane (i.e., the filtrate or permeate) is collected and used in the further purification steps. Equipment and membranes for performing such filtrations are well known in the art.

The preferred step following filtration is ion exchange chromatography on an anion exchanger. It has been shown that exchangers with Diethylaminoethyl groups give good separations, but it is believed that other anion exchangers could also be used. It is preferred that the solid support of the ion exchanger be capable of maintaining high flow rates. Sepharose has been found to be suitable for this purpose.

The preferred step following ion exchange chromatography is gel filtration chromatography. A column packing should be chosen for this step that is capable of fractionating molecules with molecular weights of less than 10,000 daltons. The preferred packing is Toyopearl® HW-40 (Rohm and Haas), but other packings well known in the art could also be used. Examples of other packings that could be used and that are commercially available are polymeric carbohydrate-based packings, e.g. Sephadex® G-10 or G-25 (Pharmacia), or polyacrylamide-based packings, e.g. Biogel® P-2, P4, P-6, P-10 or P-30 (Bio-Rad).

The preferred step following gel filtration chromatography is affinity chromatography on a boronate affinity support. These supports have been shown to be effective in the fractionation of low molecular weight compounds containing cis-diol groups. The preferred support is AffiGel® 601 (Bio-Rad). This is a boronate derivative of the polyacrylamide gel filtration support Biogel® P-6 (also available from Biorad).

The preferred mode of storage for preparations after the steps of ion exchange, gel filtration or affinity chromatography is as a lyophilized powder. The filtrate collected in the first purification step can be stored refrigerated until use. The activity of the product resulting from the purification anti-inflammatory factor can be determined using the rat paw test described above.

The results of the experiments described in Example 16 indicate that pretreatment of animals with anti-inflammatory factor preparations reduces platelet activating factor (PAF)-stimulated adhesion of neutrophils to the endothelial cells lining the venules and reduces the rate at which neutrophils emigrate from the venules. In addition, administration of factor preparations following treatment of animals with PAF was found to reduce the number of neutrophils adhering to endothelial cells. To the extent that patients or animals can benefit from these effects, the present invention encompasses the use of anti-inflammatory factor preparations. This is true regardless of the particular disease involved. Likewise, the data in Example 16 demonstrate that the anti-inflammatory factor exerts its effects on adhesion and emigration by directly interacting with cell surface CD18 antigens and preventing other molecules from interacting with this glycoprotein complex. The present invention also encompasses the use of preparations of the anti-inflammatory factor for this purpose.

As shown in Example 18, administration of a preparation of an anti-inflammatory factor to animals suppresses the host-versus-graft reaction but not the graft-versus-host reaction and causes an increase in spleen weight and the number of splenic lymphocytes. It was also shown that the lymphocyte response to concanavalin A was dependent on the preparation These data demonstrate that the anti-inflammatory factor is useful in inhibiting tissue-destructive processes and in situations where suppression of lymphocyte function is desirable.

The compositions of the present invention may be administered by any means that provides anti-inflammatory activity. For example, administration may be parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, or oral.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may, as is normal practice, also comprise additional substances other than the inert diluent. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering substances. Tablets and pills may additionally be manufactured with an enteric coating.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the pharmaceutical art. In addition to inert diluents, such compositions may also include excipients such as wetting agents, emulsifying and suspending agents and sweetening agents.

Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate.

The dosage of active ingredients in the composition of this invention can be varied; however, it is necessary that the amount of active ingredient be such that a suitable dosage form is obtained. The dosage form selected will depend on the therapeutic effect desired, the route of administration and the duration of treatment.

Dosage and frequency of administration will depend on the patient’s age and general health, taking into account the possibility of side effects. Administration will also depend on concomitant treatment with other drugs and the patient’s tolerance to the drug administered.

Having now generally described the invention, the same will be described with reference to certain specific examples which are provided herein for purposes of illustration only and which, unless otherwise indicated, are not intended to be limiting.

Example 1A (comparative example) Production of S-100 vaccine

A bacterial culture containing the spectrum of bacteria shown in Table 1 below, as obtained from the American Type Culture Collection, was cultured with 15 ml of medium and incubated overnight at 37ºC. Once good growth was obtained, approximately one-half of the bacterial suspension was used to inoculate one liter of culture broth, incubating the inoculum at 37ºC. The remaining suspension was transferred to sterile glycol tubes and stored at -20 C for up to six months.

After good growth was visible in the culture, the bacterial cells were harvested from the suspension by centrifugation for 20 minutes to remove the medium. The resulting bacterial pellet was resuspended in sterile saline and the bacterial sample was centrifuged three times to wash the medium from the cells. After the third sterile wash with saline, the bacterial pellet obtained after centrifugation was resuspended in a small amount of double distilled water.

The media-free bacterial suspension was heat-killed by placing the suspension in a glass flask in a water bath at 80ºC overnight. The viability of the culture was tested with a small amount of heat-killed bacteria. A culture solution was inoculated with heat-killed bacteria, incubated for five days at 37ºC and checked daily for growth, as the bacteria must be killed for use in the vaccine.

The heat-killed bacteria were lyophilized to dryness. The dry bacteria were then mixed with sterile saline to a concentration of 2.2 x 108 bacterial cells/ml saline (optical density of 1.0 at 660 nm).

Cows were given daily injections of 5 ml samples of the polyvalent liquid vaccine. Antibody (IgG) titer levels for the injected cattle were determined periodically by using an enzyme-linked immunoassay for bovine antibodies to the polyvalent antigen.

Example 1B (comparative example) Immunization procedures

Heat-killed bacteria were prepared in the manner described above. The resulting polyvalent antigen sample (S-100) was microencapsulated using a conventional phase separation process to produce a microparticle product containing a polyvalent antigen. Generally, the antigen-containing molded matrix materials are formed from polymers of biocompatible material, preferably biodegradable or bioerodible materials, preferably polylactic acid, polyglycolic acid, copolymers of lactic and glycolic acids, polycaptolactone, copolyoxalates, proteins such as collagen, fatty acid esters of glycerol and cellulose esters. These polymers are well known in the art and are described, for example, in U.S. 3,773,919; U.S. 3,887,699; U.S. 4,118,470; U.S. 4,076,798. The polymeric matrix material used was a biodegradable lactide-glycolide copolymer.

Heat-killed bacterial antigens are encapsulated in such matrix materials, preferably as microspheres of between 1-500 micrometers in diameter, preferably 10-250 micrometers. The encapsulation methods are conventional and include phase separation methods, interfacial reactions and physical methods. Many combinations of matrices and many concentrations of different antigens may be used to provide optimal release rates of bacterial antigens from the microparticles into the host body. These combinations can be determined by one of ordinary skill in the art without undue experimentation.

The microparticles in the example had a diameter of less than 250 micrometers. Approximately 750 mg of microparticles containing 22% (16.5 mg) of polyvalent antigen were then suspended in approximately 3 cc of a vehicle (1 wt% Tween 20 and 2 wt% carboxymethylcellulose in water).

A small group of cattle was selected from a larger herd of cattle. Five of these randomly selected cattle were chosen as controls. Four cattle were injected intramuscularly with microparticles containing polyvalent antigen. Microparticle samples were sterilized with 2.0 mRad of gamma radiation. Antibody (IgG) titer levels were determined periodically from samples of cows' milk obtained from the vaccinated cows as well as the control cows.

Example 2 (comparative example) Isolation of the MAIF factor from hyperimmunized milk Step 1: Milk filtrate production

Twenty liters of fresh milk from hyperimmunized cows were passed through a milk centrifuge (DeLaval Model 102) to remove fat.

The resulting sixteen liters of skim milk was ultrafiltered to remove high molecular weight species (over 10,000 Daltons) using a hollow fiber diafiltration/concentrator (Amicon DL-10L). The concentrator is equipped with two cartridges with a cut-off of 10,000 Dalton molecular weight (Amicon H₅P₁₀₋₄₃). The skim milk was run at a pump speed of 80 per meter and an inlet and outlet pressure of 0.207 bar and 0.172 (30 psi and 25), respectively.

Twelve liters of filtrate (< 10 000 Daltons) coming from the cartridges at a flow rate of four liters per hour was frozen or lyophilized for storage and further purification.

Step 2: Ion exchange chromatography

The anti-inflammatory factor from milk in the filtrate was first isolated using an anion exchange chromatography column.

In this procedure, DEAE-Sepharose® CL-6B gel (Pharmacia) was used to pack a 5 x 10 cm glass column equilibrated with sterile double distilled water, pH 7.0.

One liter of filtrate (< 10,000) was applied to the column and eluted with sterile double distilled water, pH 7.0, at a flow rate of 160 ml per hour. Ten milliliters of fractions were collected and monitored at 280 nm in an LKB Uvicord 4700 absorptiometer, with optical density reported on an attached recorder (Pharmacia REC-482).

Substances with positive and neutral charges other than the anti-inflammatory factor are not bound to the DEAE-Sepharose® gel. They are eluted with the through peak (first peak). The negative Charge-bearing anti-inflammatory factor is retained by the gel.

To elute the factor, the column was eluted with a stepwise gradient using sterile physiological saline, pH 7.0. A typical profile is shown in Figure 1. Bioassay of the individual fractions revealed that the second peak contains the factor. Fractions comprising the second peak and its shoulder are used for further purification. Recovery studies indicate that 8.8 grams of dried powder were obtained by this procedure.

Step 3: Gel filtration chromatography

The second peak obtained from step 2 contains the anti-inflammatory factor and other negatively charged molecules; therefore, an additional purification step was necessary. To achieve further purification, it is advantageous to use a gel filtration column to separate the different components based on molecular weight.

In this procedure, Sephadex® G-10 resin (Pharmacia) was packed into a 2.5 x 80 cm glass column and equilibrated with sterile double distilled water, pH 7.0. Two grams of the second fraction from step 2 was redissolved in sterile double distilled water and added to the top of the column. The column was eluted at the flow rate of 30 mL per hour. Fractions (3.3 mL) were collected and monitored at 254 nm and 280 nm (Pharmacia Duo Optical Unit) with the optical density reported on an attached recorder (Pharmacia REC-482).

Typically, there were 3 peaks in the elution profile, as shown in Fig. 2. The first and second peaks contained anti-inflammatory activity.

The first peak is an aggregate formed on the G-10 column containing the active factor.

The second peak contains the non-aggregated form of the factor. Both the aggregate form (peak 1) and the non-aggregated form (peak 2) are biologically active in the rat bioassay.

Example 3 (comparative example) Characterization of the anti-inflammatory factor from milk

The molecular weight of the nonaggregated form of the factor prepared by the procedure described above was found to be less than 10,000 daltons. This was inferred from the fact that the first step in isolating the factor from whey was by ultrafiltration using a membrane that did not allow the passage of species with a molecular weight > 10,000 daltons.

The factor has a negative charge. This was determined by applying the milk ultrafiltrate to a DEAE-cellulose ion exchange column. The anti-inflammatory activity did not elute from the column with water. Changing the elution medium to sodium chloride (0.9% pH) caused the elution of several peaks (Fig. 1). Neutral and positively charged species do not adhere to the ion exchange resin, and negatively charged species are eluted by increasing the salt concentration. If the permeate is less than 10,000 Daltons molecular weight When the factor was applied to the DEAE column, neutral salts and sugars eluted with water (peak 1, Fig. 1). Three characteristic peaks eluted when the buffer was changed to saline (peaks 2-4). The second peak and its shoulder contained anti-inflammatory biological activity in the rat assay. It was therefore concluded that the factor has a negative charge.

Another chemical characteristic of the factor is that it forms an aggregate during the salt removal process. This property was recognized when a permeate with a molecular weight < 10,000 daltons was passed over a Sephadex G-10 column equilibrated with double distilled water and eluted with water at pH 7 (Fig. 2). Three peaks eluted from the G-10 column; the first peak eluted with the void volume indicating a molecular weight equal to or greater than 10,000 daltons. This was unexpected since molecules greater than 10,000 daltons had previously been removed from this sample by ultrafiltration. The second peak eluted at the position expected for the anti-inflammatory factor. Both the first and second peaks demonstrated anti-inflammatory biological activity in the rat paw assay, while the third peak had no activity. It was surprising that both the first and second peaks had anti-inflammatory biological activity. The material recovered from the first peak of the G-10 column (Step 3) was lyophilized and applied to a G-100 column; a single peak was eluted with the void volume, indicating a molecular weight of 100,000 Daltons or greater. The Step 3 G-10 column simultaneously removes salt and separates the species of different molecular weights. It was therefore concluded that the anti-inflammatory factor during of passage through the G-10 column and the resulting removal of salt, a large molecular weight aggregate was formed. The degree of aggregation varied with the salt concentration.

The aggregation capacity suggests the possibility that a wide range of species with different molecular weights can be formed that possess anti-inflammatory biological activity due to the presence of the anti-inflammatory factor. The discovery of this property suggests the possibility of producing anti-inflammatory factors from milk that possess a wide range of different biochemical properties depending on the degree of aggregation of the final product. For example, preparations that possess longer or shorter biological half-lives could be prepared by using aggregates with larger or smaller molecular weights, with the molecular weight distribution controlled by the salt concentration during processing. The column chromatography procedure described herein results in the lowest molecular weight species obtained with biological activity (i.e., peak 2 from step 3 G-10 column). This observation also suggests using other methods to form the aggregates. For example, dilution in water causes aggregation to occur. Chemical agents that bind salts, particularly calcium, can cause the formation of the aggregate. Having made this discovery, other methods to form and separate the aggregates will be apparent to those of ordinary skill in the art.

Example 4 (comparative example) Biological activity assay

The anti-inflammatory effect of purified anti-inflammatory factor was tested on edema induced by injecting a solution of carrageenan into the paws of rats. A lyophilized sample of the anti-inflammatory factor milk preparation was dissolved in the appropriate vehicle and given intraperitoneally to laboratory rats. The carrageenan was then administered to the rats in an amount of 0.1 ml of a 1% saline solution in each hind paw. The paws were measured using a thickness gauge before the injections and 2.5 hours after the injections. The results are illustrated in Tables 2 and 3. In these tables, the abbreviation MAIF refers to the anti-inflammatory factor milk preparation obtained using the procedures described above in Examples 1 and 2.

The nonaggregated form of the factor (peak 2 from the G-10 column) from control and hyperimmune milk caused a reduction in inflammation of the rat foot at doses ranging from 1 mg to 0.25 mg (Table 2). Both hyperimmune milk and normal milk showed activity; however, the hyperimmune material was more potent. We concluded that the anti-inflammatory factor was present in greater concentration in the milk of hyperimmune cows.

The second peak from the DEAE column showed activity when isolated from either hyperimmune milk or regular milk. The activity is substantially greater in the hyperimmune milk (Table 3).

The first peak from the G-10 column, which is the aggregated form of the factor, showed activity in rat paw tests (Table 2). However, the aggregated form is not as potent as the nonaggregated form on an equal weight basis.

It is concluded from these studies that the anti-inflammatory factor occurs naturally in cow's milk. Hyperimmunization of cows causes a higher concentration of the factor in the milk. The factor is a small, negatively charged molecule that can be separated from milk by a variety of methods. The factor can form large molecular weight aggregates that do not occur naturally in milk but form during processing.

Example 5 (comparative example) Chemical analysis of the anti-inflammatory factor

Samples of the anti-inflammatory factor were chemically analyzed. As determined by X-ray diffraction studies, the factor has a non-crystalline structure. MAIF preparations gave an elemental analysis consistent with a carbohydrate composition. The C, H, O ratios were consistent with a polymeric or oligomeric material with some carbinol groups oxidized to carboxyl. The slight excess of calcium equivalents over chloride ions can be partially explained by carboxylate salts. The remainder may be sodium or potassium salts. However, the melting behavior, or rather the non-melting behavior, suggested salt-like and/or higher molecular weight compositions. The material contains in the present State of purity obviously contains a variable amount of salts of calcium and chloride, probably CaCl2.

No preparation contained a significant amount of nitrogen, which precludes a peptide component in its composition. Likewise, the lack of significant nitrogen may preclude the presence of amino sugars and other nitrogen-containing materials such as various complex lipids as the major component(s).

Pyrolytic mass spectra showed significant traces of 18 carbon fatty acids. This fact, together with traces of N and P, suggests the presence of a complex lipid in the preparation.

Infrared spectroscopy showed absorptions consistent with carbinol and carboxylate functionalities. UV, visible and fluorescent spectroscopy did not show any significant amount of chromophores in addition to those already revealed in the infrared region.

The chemical studies are consistent with an oligomeric carbohydrate, with the carbonyl function (aldehyde or ketone) bound in the linkages of the subunits. The oligomeric carbohydrate also contains some side chain oxidation to carboxylate.

The MAIF preparation is essentially, but not completely, pure.

Example 6 (comparative example) Rat paw edema tests: Oral administration

The rat carrageenan paw assay was used to test the efficacy of anti-inflammatory factor as an in vivo anti-inflammatory agent. Thirty adult white rats were randomly divided into three groups of ten rats per group. The groups each received 10 mg of skim milk powder from hyperimmunized animals, 10 mg of skim milk powder from nonimmunized animals, or no treatment (only 20 ml of water per day) for five consecutive daily treatments. The powders were administered orally in 20 ml of water. On the fifth day, 0.1 ml of 1% carrageenan in saline was injected into the right paw of each rat. This procedure is known to cause acute inflammation (edema). Twenty-four hours after injection, the rats were sacrificed, the paws amputated, and the weights of the left (control) and right (edematous) paws were compared. The results of the assay are shown in Table 4 (expressed as weight in grams) and in Fig. 3 (expressed as a percentage of the average weight of the control paws).

The inflammatory response to a carrageenan injection was significantly reduced in the rats treated with immune milk compared to the control groups with non-immune milk and water. No evidence of No adverse effects or adverse effects on the general health of the rats were detected. From these data, it can be concluded that daily consumption of skim milk powder by hyperimmunized animals almost completely blocks the inflammatory response induced by carrageenan injection into the paw of rats.

Example 7 (comparative example) Quantitative rat paw edema tests

A series of experiments were conducted using the hyperimmune milk fraction. The experiments were designed to confirm the anti-inflammatory activity of the milk anti-inflammatory factor when administered intraperitoneally, to establish a dose-response curve, to explore alternative routes of administration, and to investigate dosage ranges that could form the basis of further studies.

Peak I from the G-10 column supplied by Stolle Milk Biologics International was prepared according to the procedures described in U.S. Patent No. 4,956,349. Lactose obtained from commercial sources was used as a placebo. Aspirin® was used as a positive control. Aspirin® was dissolved in water and given orally by gavage at a rate of 200 mg per kilogram, a dose known to be active in the assay. A 2% solution of kappa-carrageenan (Sigma C-1263) was shown to produce the most reproducible results and was used in these experiments. The paw assay was modified by using isotopically labeled human serum albumin (125I-HSA), which localizes in the carrageenan-induced lesion in direct proportion to the volume of exudate. By reducing the total radioactive By determining the number of counts in the paw and comparing these to the counts in a known volume of plasma from the injected animal, a direct measurement of edema in microliters of plasma equivalents is obtained. 125I-HSA was injected intravenously at a dose of 1.0 microcurie per rat. Female dark agouti rats were used. Rats were approximately 12 weeks old, weighed between 160 grams and 200 grams, and were obtained from the inbred colony.

To perform the carrageenan paw assay, 0.1 ml of 2% carrageenan was injected subcutaneously into each hind paw of an anesthetized rat. This injection was immediately followed by an injection of 1.0 microcurie of 125I-HSA in 0.5 ml of saline into the tail vein. After four hours, each rat was weighed, blood samples were obtained, and the rats were euthanized. Both hind feet were then removed and the levels of radioactivity in each foot and in the 200 μl plasma standard were measured in a fully automated gamma counter. From these measurements, the volume of edema in each foot was calculated and expressed in microliters.

Experiment 1: Response to an intraperitoneal dose

Fig. 4 illustrates the effect of intraperitoneal administration of a purified preparation of MAIF compared with lactose (CON), Aspirin® and no treatment (No Rx). All treatments (lactose, Aspirin®, MAIF) were given 30 minutes before the injection of carrageenan.

The carrageenan injection resulted in an edema of on average 250 ul (No Rx). The edema was inhibited by aspirin and all doses of the MAIF preparation, but was but not inhibited by lactose. The response curve obtained with the MAIF preparation to an intraperitoneal dose, which was obtained by expressing the data as a percentage of the edema of the average control (no treatment), is shown in Fig. 5.

Experiment 2: Effects of different routes of MAIF administration

Fig. 6 illustrates the effect on paw edema of administering lactose and a preparation of purified MAIF orally (ORAL), intramuscularly (IM), subcutaneously (SUB Q) and intravenously (IV). Also shown are a positive control (Aspirin®) and a non-treated control (NO Rx).

The preparations were administered before carrageenan challenge according to the following schedule:

Aspirin®: orally, 30 minutes before;

MAIF subcutaneous: 1 hour before;

MAIF orally: 24, 16 and 1 hour before;

MAIF intramuscularly: 30 minutes before;

MAIF intravenously: at the time of irritation (isotope was also injected).

The results indicate that, expressed as a percentage of average control edema in each separate assay, the anti-inflammatory factor inhibited edema formation in all routes of administration. Forty milligrams of the MAIF preparation given intravenously completely abolished the inflammatory response to carrageenan. These results demonstrate the anti-inflammatory activity of MAIF and, in view of the results of Experiment 1 above, suggest that the Order of efficacy for different routes of administration is as follows: IV > IP > IM > SUB Q > ORAL.

Experiment 3: Effect on edema after intravenous and extended oral administration: dose response

Figure 7 shows the effects of IV and oral administration of a purified preparation of anti-inflammatory factor on paw edema in rats. Oral MAIF treatment (40 mg per rat per day) was given daily for six days and also one hour before carrageenan challenge (PO). Intravenous treatments (5, 10, 20 mg) were given at the time of carrageenan challenge (IV). A positive control (Aspirin®) and a negative control (no treatment) are also shown.

The results shown in Fig. 7 demonstrate that all three doses of the MAIF preparation result in anti-inflammatory activity that even exceeds the activity of aspirin in the assay, while prolonged oral administration results in pronounced but limited activity.

The study was therefore extended to test the effects of further reduced intravenous doses of anti-inflammatory factor. Intravenous doses of lactose placebo were included as a control. The results of these studies are shown in Figure 8. Intravenous doses of 2.5 and 1 mg of the MAIF preparation (IV) induced anti-inflammatory activity in the range of that induced by aspirin. Ten ml of intravenous lactose placebo (10 mg PLAC IV) did not induce activity in this range.

An intravenous dose response curve was obtained by combining the results from Experiments 2 and 3 and expressing these results as a percentage of average control edema (no treatment) in each separate assay. The curve is shown in Fig. 9.

The conclusions drawn from the quantitative rat paw edema tests are as follows: The milk fraction peak I from the G-10 column, extracted and purified as described in U.S. Patent No. 4,956,349, consistently demonstrates anti-inflammatory activity when tested in the rat paw edema model. An intravenous dose of 4 mg of MAIF preparation per rat at the time of carrageenan injection is sufficient to satisfactorily inhibit edema and was therefore chosen as a standard against which other preparations will be compared in further experiments.

Example 8 (comparative example) Anti-inflammatory properties of preparations of hyperimmune milk obtained from identical twin cows

The effect of vaccination on the anti-inflammatory activity of milk was studied by testing the bioactivity of different milk fractions obtained from identical twin cows. Based on the extraction methods described in US Patent No. 4,956,349, an extraction scheme was established using ultrafiltration. The sequence of operations was as follows:

Milk samples were prepared from immunized twin cows, non-immunized control twin cows, and reconstituted skim milk powder previously prepared from immunized cows. The sample group consisted of 45 sets of identical twin cows. One cow from each twin set was vaccinated twice a week with Stolle 5100 mixed bacterin (described in U.S. Patent No. 4,956,349). The bioactivity of the various fractions was tested by intravenous injection using the rat carrageenan paw assay described above.

The hypotheses to be tested were that

(a) hyperimmunization was responsible for the anti-inflammatory activity described above.

(b) MAIF could be extracted on a commercial scale by ultrafiltration, and

(c) dilution of the permeate would cause aggregation of the anti-inflammatory factor, causing it to be retained by the 30,000 molecular weight ultrafiltration membrane.

Figure 10 illustrates the results of a twin herd ultrafiltration experiment designed to test the bioactivity of various fractions prepared from milk from non-vaccinated control twins and from reconstituted milk powder from immunized cows. The fractions tested are as follows: Peak I, G-10 column preparation, 4 ml (OHIO MAIF STD); final R2 retentate from non-vaccinated twin (CONTROL TWIN R2); final P2 permeate from the reconstituted milk powder (RECON S100 P2); dialyzed final R2 retentate from non-vaccinated twin (CON DIALYZED R2); dialyzed final retentate from the reconstituted milk powder (S100 DIALYZED R₂).

No anti-inflammatory activity could be detected in the final R2 retentate fraction obtained from non-immunized cows, even after dialysis. No anti-inflammatory activity was detected in the final permeate P2 fraction prepared from the reconstituted milk powder. The reconstituted milk powder retentate R2 fraction, following dialysis, showed anti-inflammatory activity in the range of the MAIF standard activity.

Fig. 11 illustrates the results of double-herd ultrafiltration experiments designed to test the bioactivity of different milk fractions obtained from vaccinated and unvaccinated twin cows and reconstituted milk powder from immunized cows. The fractions tested are as follows: Peak I, G-10 column preparation, 4 ml (OHIO MAIF STD); dialyzed final retentate R2 from unvaccinated twins (CON DIALYZED R2); final retentate R2 from the reconstituted milk powder (RECON S100 R2); the final retentate R2 from vaccinated twins (IMMUNE TWIN R2); first retentate R1 from the reconstituted milk powder diluted for: 1 (S100 DILUTED R1).

A small amount of anti-inflammatory activity was detected in the dialyzed retentate R2 from non-vaccinated control twins or in the non-dialyzed retentate R2 from the vaccinated twins. Some activity is evident from the scattergram. The R2 retentate prepared without dialysis from reconstituted Stolle milk powder from immunized cows was potently anti-inflammatory. However, the preparation prepared by diluting the reconstituted milk prior to ultrafiltration was only slightly active, in contrast to diluting whey prepared from the milk. This result indicates that anti-inflammatory activity is more efficiently extracted from the whey fraction.

Figure 12 illustrates the results of double-herd ultrafiltration experiments designed to test the bioactivity of a dialyzed retentate from vaccinated twin cows. The fractions tested are as follows: Peak I, G-10 column preparation (OHIO MAIF STD); dialyzed final retentate R2 from vaccinated twins (IMM DIALYZED R2); dialyzed final retentate from the G-10 preparation (DIALYZED OHIO MAIF). The results show that anti-inflammatory activity was present in the R2 fraction from the immunized twin after dialysis. Dialyzed MAIF was more active in the assay than the nondialyzed MAIF standard. This result suggests that dialysis is an effective means to further concentrate the milk factor responsible for anti-inflammatory activity.

The above results presented in Figures 10-12 support the following conclusions: (1) Anti-inflammatory activity can be extracted from reconstituted milk of immunized cows by ultrafiltration of the diluted permeate. (2) Anti-inflammatory activity was not demonstrated in the above preparations prepared from the milk of non-immunized cows. (3) Anti-inflammatory activity was demonstrated in the final retentate R2 after ultrafiltration of diluted permeate prepared from the milk of immunized cows, however dialysis was necessary to demonstrate activity.

Example 9 (comparative example) Stability of MAIF, heating and proteinase treatment of MAIF

The previous evidence that the anti-inflammatory factor was not chemically a protein or peptide was based primarily on chemical analyses which consistently showed an almost complete absence of nitrogen. For further characterization of the anti-inflammatory factor, several preparations were tested in the rat paw edema assay using 4 mg of Peak I, G-10 column preparation intravenously as a standard. The following treatments were administered: proteinase (pronase) treatment for six hours; six hours no proteinase treatment control; untreated positive control; heating at 100ºC for 30 minutes.

The results of this assay are illustrated in Fig. 13. The conclusions drawn from this study were that the anti-inflammatory activity was not due to a protein or peptide and that the anti-inflammatory factor was not inactivated by cooking. The effectiveness of pronase treatment was verified by the discovery that the parallel pronase treatment completely denatured the milk protein.

Example 10 (comparative example) Anti-inflammatory activity of further purified MAIF and whey protein concentrate from immunized cows

Retentate and permeate from ultrafiltration using an Amicon YM5 membrane were tested for biological activity using intravenous administration in the rat paw edema assay. In this procedure, the MAIF in Peak I from the G-10 column, prepared according to U.S. Patent No. 4,956,349, was further purified by ultrafiltration on an Amicon YM5 membrane. This membrane retains molecules with a molecular weight of 5000 or greater. Whey protein concentrates (WPCs) were also prepared from milk of immunized animals and filtered through the YM5 membrane. The following samples were tested in the assay using 4 mg of Peak I, G-10 column preparation intravenously as a standard: permeate from Amicon YM5 ultrafiltration; retentate from Amicon YM5 ultrafiltration; WPC from immunized cows, 30 mg per rat; WPC from commercial production (non-immunized cows), 30 mg per rat.

The results of this assay are illustrated in Figure 14. From these results it is clear that all of the activity is in the retentate, which represents approximately 0.5% of the total weight of the fraction applied to the YM5 filter. The reduction in edema seen in this experiment was achieved after administration of 20-25 micrograms of material.

Regarding the activity of WPC, WPC produced from hyperimmunized animals showed anti-inflammatory activity as expected. Interestingly, WPC produced from non-immunized animals also showed anti-inflammatory activity. The presence of anti-inflammatory activity in the milk of non-immunized cows is not surprising since it must be a natural substance. The detection reflects the sensitivity of the bioassay.

Example 11 (comparative example) Continuous monitoring of carrageenan-induced paw edema

It was found that 4 mg of MAIF preparation, given intravenously at the time of carrageenan injection, reduced the accumulation of edema in the paw by between 40% and 50%. Although these results provided evidence that the material contained an anti-inflammatory moiety, there was little evidence regarding the site of action or the pharmacological profile of MAIF. To obtain such data, it was necessary to establish a procedure that allowed the continuous monitoring of paw edema by the response to carrageenan. This was accomplished by placing the rat foot in a dismantled gamma ray detector.

The procedure required that the animals be anesthetized for up to four hours and, since anesthetics are known to suppress the inflammatory response, it was first necessary to determine the effect of anesthetics on carrageenan-induced edema. Five agents commonly used to anesthetize rats were therefore tested; these were ether, chloral hydrate, Innovar-vet, Nembutal and urethane. The results are shown in Fig. 15.

From these results it was clear that ether was the anesthetic of choice when the inflammatory response was to be evaluated by this method. The shape of the curve obtained using ether indicated a biphasic response. To describe the response in more detail, another experiment was performed in which the volume of edema was measured at 12 time points over a period of 5 hours. The results confirmed a biphasic response. The early response came between 0 and 1 hour after stimulus and the later phase response came between 1.5 and 2 hours (Fig. 16).

The two phases, which have also been observed by other researchers, have been called the non-phagocytic inflammatory response (NPIR) and the phagocytic inflammatory response (PIR), respectively.

The NPIR is initiated in response to injury by soluble mediators such as histamine and bradykinin, whereas the PIR depends on the involvement of neutrophils. The protocol was therefore to administer MAIF and continuously monitor the accumulation of edema to determine whether the anti-inflammatory property of the agent was a result of an effect on the early non-cellular (NPIR) or the later cellular (PIR) phase. 5 mg or 40 mg of MAIF preparation was administered intravenously per rat at the time of carrageenan challenge and the accumulation of edema was monitored at regular intervals over a four hour period. Neither dose affected the accumulation of edema during any phase (Fig. 17).

This result was surprising since many previous analyses determining the effect of purified preparations of MAIF on edema 4 hours after carrageenan-induced irritation had demonstrated significant anti-inflammatory activity in the fractions. It was therefore likely that continuous exposure to ether suppressed or inactivated the active anti-inflammatory component of MAIF in vivo.

Previous studies indicated that short-term exposure to ether does not affect the activity of the anti-inflammatory factor. Therefore, an experiment was conducted in which the effect of MAIF on progressive edema accumulation was determined at only four time points, 0, 1, 3 and 4 hours, in order to limit the exposure of the animals to ether. The measurement point at 1 hour was chosen to estimate the effect on the early non-phagocytic inflammatory response, while the measurement points at 3 and 4 hours were chosen to quantify the effect on the later phagocytic inflammatory response. The 40 mg MAIF preparation administered in this experiment resulted in a reduction in the accumulation of edema during the secondary, phagocytic cell-driven phase, but had no significant effect on the primary, soluble mediator-driven phase (Fig. 18).

The following conclusions can be drawn from these series of experiments.

1. Ether is the preferred anesthetic for use in experiments where the inflammatory response to carrageenan is to be continuously monitored.

2. Continuous ether anesthesia inhibits the in vivo anti-inflammatory activity of anti-inflammatory factor in the carrageenan paw assay.

3. MAIF improves inflammation by inhibiting the late, phagocytic cell-mediated phase of the inflammatory response to carrageenan.

Example 12 (comparative example) Time course of the effect of MAIF on carrageenan-induced paw edema

Additional trials were conducted in which the agent was administered at selected times before or after carrageenan injection rather than at the time of irritation. The purpose of the study was to provide information regarding:

(a) the most effective timing for administration of MAIF in relation to the inflammatory stimulation;

(b) the biological half-life of the anti-inflammatory moiety;

(c) the points in the development of the inflammatory response that are influenced by MAIF.

The study was conducted in three parts. A preparation of MAIF was administered intravenously at a dose of 4 mg/rat at one of 11 time points ranging from 150 minutes before to 150 minutes after an injection of carrageenan. The results of this experiment are shown in Fig. 19 and Table 5.

Significant inhibition of edema was observed at all time points examined; however, the level of inhibition was lower at the outer extremes (±150 min). An interesting cyclic response to MAIF administration was observed in the groups treated closer to the time of challenge. The fact that MAIF was more effective when given 30 minutes after challenge than when given 15 minutes after challenge supports the concept that the secondary, phagocytic cell-related phase of the response is inhibited by the agent. The preparation of MAIF strongly inhibited the response to carrageenan when administered 15 minutes before or at the time of challenge. It is also evident that the agent has a relatively long half-life in serum (1-2 h) and its efficacy is related to the time of challenge and the dynamic nature of the inflammatory response.

It is therefore assumed that the anti-inflammatory effect is due to an effect on inflammatory cells, probably neutrophil leukocytes.

Example 13 (comparative example) Effect of MAIF on the reverse passive Arthus reaction

The possibility that the anti-inflammatory factor could influence neutrophil involvement was investigated by evaluating the ability of the material to modulate the reverse passive Arthus reaction (RPA). This immune complex-induced response is primarily neutrophil-mediated, and agents that influence the development of the response do so via an effect on these cells. To induce RPA, rats were injected intradermally with rabbit antibody to ovalbumin and intravenously with native ovalbumin. Ovalbumin/ovalbumin antibody immune complexes formed in and around the skin blood vessel walls, neutrophil leukocytes bound to the Fc portion of the antibody, and a vigorous inflammatory response was initiated. It should be noted that although the response is initiated by immune complexes, it occurs independently of the host immune system.

Three parameters are used to quantify RPA. These are,

(1) Edema - measured using the accumulation of ¹²⁵I-HSA,

(2) Bleeding - estimated by in vivo pre-labelling of RBC's with ⊕5;⊕9Fe and

(3) neutrophil accumulation – measured by determining tissue levels of the neutrophil-specific enzyme myeloperoxidase (MPO).

These assays are known to the person of ordinary skill in the art.

Eighteen rats were divided into three groups of six. At four sites on the back of each animal, rabbit anti-ovalbumin (40 μl) was injected intradermally and immediately thereafter 2 mg of ovalbumin was injected intravenously. One group of animals received no other treatment and served as controls. The second group was injected intravenously with 20 mg of a lactose preparation, while the last group was injected intravenously with 20 mg of a purified preparation of MAIF. Both the lactose and MAIF preparation were administered with the ovalbumin. The magnitude of the response was estimated 3.5 hours after challenge. When the MAIF preparation was administered intravenously at a dose of 20 mg/rat before initiation of the RPA response, there was a highly significant inhibition of the three parameters used to measure the response (Table 6, Fig. 20). The lactose control material also caused a small and insignificant suppression of neutrophil accumulation and bleeding, indicating that there is a small amount of anti-inflammatory activity in normal milk.

Since neutrophils are the primary mediators of RPA, these results provided additional evidence that MAIF was able to inhibit the inflammatory response via an effect on neutrophil function.

Example 14 (comparative example) Effect of MAIF on neutrophil migration

To actually participate in an inflammatory response, neutrophils must first migrate from the vasculature to the site of inflammation. To determine whether the anti-inflammatory factor affects neutrophil migration, a model of inflammation was used that involved the subcutaneous implantation of sterile polyurethane sponges. The sponges are removed at intervals after implantation, and by weighing the sponges and then extracting and counting the cells in the infiltrate, both the fluid and cellular phases of the response can be quantified. Twenty-four hours after implantation, >95% of the cells found in the sponge were neutrophils.

Two experiments were conducted. In the first, animals were treated with either 5, 10, 20 or 40 mg of a purified MAIF preparation at the time of sponge implantation. Sponges were removed 24 hours after implantation. Each group consisted of between 5 and 8 rats and two sponges were implanted into each animal. The results are shown in Fig. 21.

Twenty or 40 mg of MAIF preparation administered intravenously at the time of sponge implantation had a marked effect on the ability of inflammatory cells to migrate. A less pronounced but equally significant inhibition of fluid accumulation was also observed. The two lower doses of MAIF had no demonstrable effect in this inflammatory model.

A second experiment designed to demonstrate the time-dependent relationship between inflammatory challenge (sponge implantation) and MAIF administration was conducted. In this study, 20 mg of MAIF preparation was administered intravenously 30, 60, or 120 minutes after sponge implantation. A fourth control group was left untreated. There were five animals in each group. Two sponges were implanted in each animal and these were removed after 24 hours. The results are illustrated in Fig. 22. Included in this graph are results obtained from a sample group of rats that received 20 mg of MAIF preparation at the time of implantation (see Fig. 21).

The results of the time course of the effect of MAIF on carrageenan-induced paw edema show that MAIF is relatively ineffective when administered 60 minutes or later after the challenge. It is noteworthy that while 20 mg of the MAIF preparation is necessary to suppress the inflammation associated with the sponge implantation, 4 mg was sufficient to inhibit the carrageenan-induced edema. Without being bound to this interpretation, this difference can be related to the different levels of challenge presented to the host. The sponge implant is a relatively benign stimulus which induces a slow inflammatory response and the majority of cells accumulate between 8 and 16 hours after implantation (Fig. 23). On the other hand, subcutaneous injection of carrageenan is a very powerful stimulant which induces a correspondingly strong response over a relatively short period of time (Fig. 16).

Example 15 Alternative method for purifying the anti-inflammatory factor from milk (preparation "AIF")

The following example describes a method for purifying anti-inflammatory factor from milk in unaggregated form at its lowest molecular weight. The preparation resulting from the purification steps described herein was given the designation "AlF" to distinguish it from the preparation obtained using the procedure described in Example 2. In the present example and the subsequent example (i.e., Example 16), the active factor within the preparations is referred to simply as the "anti-inflammatory factor." All purification steps were carried out in such a way as to minimize possible contamination with bacteria or pyrogens. Sterile water was used used to prepare solutions and all glassware was depyrogenated.

Step 1: < 10 00 molecular weight ("MW") Ultrafiltration

Fresh S100 immune skim milk (see Example 1 for a description of the procedures used to obtain the immune milk) was pumped at 30 psi through an ultrafiltration membrane with a MW cut-off of 10,000 (Filtron). The permeate was collected in depyrogenated bottles kept on ice. The permeate was sterile filtered and refrigerated until use. The MW < 10,000 permeate contains the anti-inflammatory factor from milk and also low molecular weight peptides, oligosaccharides and a large amount of lactose. Anti-inflammatory activity in the permeate occurred in an unaggregated low molecular weight form.

Step 2: DEAE-Sepharose® chromatography

An initial fractionation of anti-inflammatory activity was performed on DEAE-Sepharose®. A 5 x 50 cm column containing one liter of DEAE Sepharose was equilibrated with permeate buffer. The permeate buffer is a sterile, endotoxin-free solution containing the diffusible ions found at the corresponding concentrations in bulk milk. The permeate buffer contains CaCl2, MgCl2, NaCl, Na citrate and NaH2PO4. Typically, approximately eight liters of MW < 10,000 permeate is pumped over the DEAE-Sepharose® column at a flow rate of approximately 500 ml per hour. The column eluate was monitored at 280 nm. The column was washed with distilled water until the absorbance returned to baseline at 280 (there were typically approximately 6 to 8 liters of distilled water is required). Anti-inflammatory activity was bound to the column and was eluted with approximately 4 liters of 0.5 M ammonium acetate in water, pH 7.4. The eluate was lyophilized to dryness and weighed. The weight of recovered material obtained from eight liters of permeate was typically between 15 and 20 grams. Since ammonium acetate evaporates completely during lyophilization, the residual weight represents the weight of bound material. Anti-inflammatory activity was tested in the mouse neutrophil migration inhibition assay.

Step 3: H-40 Chromatography

The material eluted from the DEAE-Sepharose® column was further fractionated on a size-sizing column to separate the factor responsible for anti-inflammatory activity from other low molecular weight components. Eight grams of the DEAE sample was dissolved in 50 ml of distilled water and applied to a 2.5 x 150 cm column containing 736 ml of Toyopearl® HW-40 (Rohm and Haas) equilibrated with water. The column was developed in distilled water at a flow rate of 40 ml per hour and the eluate was monitored at 280 nm. Fractions were collected and tested for anti-inflammatory activity in the mouse neutrophil migration inhibition assay. Fractions demonstrating activity and minimal absorbance at 280 nm were pooled and lyophilized. Approximately 80 mg of material containing anti-inflammatory activity was recovered from eight liters of permeate.

Step 4: AffiGel 601 Chromatography

The final purification step involved affinity chromatography of the active factor in a column packed with a boronate-derivatized polyacrylamide-based medium (AffiGel® 601, Bio-Rad) that has an affinity for coplanar adjacent cis-hydroxyl groups. Forty mg of material derived from low molecular weight HW-40 was equilibrated in 10 mL of 0.25 M ammonium acetate, pH 7.0, and applied to the AffiGel® column, which had also been equilibrated in 0.25 M ammonium acetate. The eluate was monitored at 280 nm. The column was washed with 400 mL of 0.25 M ammonium acetate at a flow rate of 50 mL per hour until the absorbance at 280 nm returned to baseline. The AffiGel® column was then eluted with 1600 ml of 0.1 M formic acid, pH 2.8. The eluate was tested for activity in the mouse neutrophil migration inhibition assay and lyophilized to dryness. Approximately 8 to 10 mg of bound material containing anti-inflammatory activity was recovered from 8 liters of permeate.

The preparation obtained by this procedure is designated "AlF". The preparation was highly purified for anti-inflammatory factor, but is not homogeneous. The preparation exhibited anti-inflammatory activity in the mouse neutrophil migration inhibition assay, rat paw edema assay, rat ear swelling assay, and blocked the binding of neutrophil leukocytes to rat mesenteric venous endothelium (visualized by intravital microscopy). Based on comparative analyses in the mouse neutrophil migration inhibition assay, AlF is approximately 55,000 times purer than the original skim milk permeate with MW < 10,000.

Example 16 Effect of anti-inflammatory factor preparations on the adhesion of neutrophils to endothelial cells and on the emigration of neutrophils from the vascular supply

The effect of the anti-inflammatory factor on the adhesion of neutrophils to endothelial cells and on the emigration of neutrophils from the vasculature was tested. Two different preparations of the anti-inflammatory factor were used. One preparation was prepared using the purification procedure described in Example 2. For the purposes of the present example, this preparation is referred to simply as "MAIF". The other preparation of the anti-inflammatory factor was prepared using the purification procedure described in Example 15 and is referred to as "AlF" in both that example and the present example. It is understood that both MAIF and AlF contain the anti-inflammatory factor in different states of purity.

Chemicals:

Human serum albumin, trypsin, platelet activating factor (PAF), phorbol myristate acetate (PMA), propidium iodide, and Histopaque were obtained from Sigma Chemicals Co., St. Louis, Mo. Human neutrophil elastase was purchased from Calbiochem. A murine anti-human CD18 monoclonal antibody (IgG1 subclass; FITC conjugate) and a murine anti-keyhole limpet hemocyanin (IgG1 subclass; FITC conjugate), used as a negative control antibody, were purchased from Becton Dickinson Systems Inc., Mountain View, CA. Simply CellulareTM Microbeads were purchased from Flow Cytometry Standards Corp., Research Triangle Park, NC. The other reagents were of the best commercially available purity and were used without further purification.

In vivo procedures: Intravital microscopy.

Twenty-four male Wistar rats (180-250 g) were maintained on a purified laboratory diet and fasted for 24 hours prior to surgery. Animals were initially anesthetized with pentobarbital (12 mg/100 g body weight). A right carotid artery and jugular vein were cannulated to measure systemic arterial pressure (Statham P23A pressure transducer and a Grass physiological recorder) and drug administration, respectively. A medial abdominal incision was made and animals were positioned supine. A segment of the medial jejunum was advanced through the abdominal incision and all exposed tissue was covered with saline-soaked gauze to minimize tissue dehydration. The mesentery was carefully positioned over an optically clear viewing pedestal that allowed fluoroscopy of a 2 cm2 segment of tissue. The temperature of the socket was maintained at 37ºC using a constant temperature circulator (Fisher Scientific, Model 80). Rectal and mesenteric temperatures were monitored using an electrothermometer. The mesentery was wetted with warmed bicarbonate buffered saline (pH 7.4). An intravital microscope (Nikon Optiphot-2, Japan) with a X25 objective lens (Leitz Wetzlar L25/0.35, Germany) and a X10 eyepiece was used to observe the mesenteric microcirculation. A video camera mounted on the microscope projected the image onto a color monitor and the images were recorded using a video recorder. for playback analysis. Single unbranched venules with diameters between 25 and 40 µm were selected for study. Venule diameter was measured online using a video caliper. The number of adherent and emigrated neutrophils was determined off-line while the videotape was playing. A neutrophil was considered to be adherent to the venule endothelium if it remained stationary for 30 seconds or more. Rolling neutrophils were defined as those with white blood cells moving at a speed slower than that of red blood cells in the same vessel. The speed of rolling leukocytes was determined by the time it took a leukocyte to traverse a given distance along the length of the venule.

Experimental protocol.

After all hemodynamic parameters were stable, images of the mesentery were recorded for 5 minutes. The mesentery was then superfused (iv.) for 60 minutes with 100 nM PAF in the presence of either 40 or 5 mg/rat of the MAIF preparation. Measurements of the above parameters were again performed at 30 and 60 min from PAF superfusion. In two experimental groups, the mesenteric preparations were re-exposed to PAF as described above, but received either 40 or 5 mg/rat of the MAIF preparation at 30 minutes. In three additional experiments, the AlF preparation was given either as a pretreatment or as a posttreatment.

In vitro procedures Isolation of neutrophil leukocytes.

Neutrophil leukocytes from healthy donors were purified by dextran sedimentation followed by hypotonic Lysis and Histopaque centrifugation. Except for the dextran sedimentation step, which was performed at room temperature, cells were maintained at 4°C during the isolation procedure. Cell preparations contained 95% neutrophils and more than 99% of these were viable as determined by using Trypan Blue. After isolation, neutrophils were resuspended in phosphate buffered saline (PBS) at a final concentration of 2 x 106 cells/ml. Aliquots of cells were then incubated at 37°C for 20 minutes with varying concentrations of either the MAIF or the AlF preparation. After washing, neutrophils were incubated in the dark at 4ºC for 30 minutes with saturating concentrations of fluorescein-conjugated murine anti-human CD18, human CD11b, IGG coated microbeads (Simply CellulareTM Microbeads) or the murine negative control antibody.

Immunofluorescence staining and FACS analysis.

Direct immunofluorescence as a measure of CD18 surface expression was determined by analysis on a FAC scan (Becton Dickinson Systems Inc., Mountain View, CA) using the channel number (log scale) representing the mean fluorescence intensity of 10,000 cells. Logarithmic channel numbers were converted to linear values using methods well known in the art. The specific mean fluorescence intensity for cells stained with CD18 antibodies was calculated after subtraction of the mean fluorescence intensity of cells exposed to the negative control antibody. Nonviable cells were sorted using propidium iodide.

Superoxide assay.

Superoxide production from isolated neutrophils was measured following PMA and N-formyl-Met-Leu-Phe ("fMLP") stimulation in the presence of various concentrations of MAIF. The reduction of cytochrome c by activated neutrophils was measured using a spectrophotometer (Hitachi U2000) at 550 nm. Briefly, the sample was placed in two cuvettes and one cuvette was used as a reference. The latter contained superoxide dismutase (superoxide scavenger). Neutrophils were allowed to equilibrate at 37ºC for 5 min in the presence of various concentrations of MAIF and the cells were then stimulated with either PMA or fMLP. Superoxide production was measured for 3 min.

Protease release.

Albumin labeled with 125I was coated on wells and allowed to dry overnight. Unbound albumin was washed and then PMA-stimulated neutrophils were incubated in the wells for 1 hour in the presence or absence of various concentrations of MAIF. The free radioactivity within the supernatant of the wells was divided by the total radioactivity within each well to estimate the level of proteolysis.

Results:

The results are summarized in Fig. 24-30 and Tables 7-9. Fig. 24 demonstrates that PAF superfusion increased neutrophil adhesion to postcapillary venules by approximately 6-fold over a 60-minute period. 40 mg/rat of the MAIF preparation reduced PAF-induced neutrophil adhesion by more than 90% at 30 minutes and by more than 80% at 60 minutes. Interestingly, MAIF pretreatment also appeared to increase the number of adhesive neutrophil leukocytes. prior to PAF exposure. The lower concentration of MAIE (5 mg per rat) was less effective in reducing leukocyte adhesion by 50% at 60 min. The AlF preparation at a concentration of 0.01 mg per rat showed a reduction in leukocyte adhesion of approximately 50% at 60 min. At a ten-fold higher concentration of AlF, a very large increase in leukocyte adhesion was observed (data not shown). The adhesion was so dramatic that the videotape could not be analyzed. Fig. 25 shows the effect of MAIF and AlF on neutrophil emigration. MAIE at a concentration of 40 mg per rat and 5 mg per rat and AlF at a concentration of 0.01 mg per rat were found to completely prevent the increase in neutrophil emigration with time of PAF exposure. The neutrophil flux showed no significant change in the MAIE-treated group compared with the untreated group (Fig. 26). When AlF was given, we initially observed more neutrophil rolls than usual, but the number decreased with time.

In a second series of experiments, the various anti-inflammatory agents were administered after neutrophils had already adhered (Fig. 27). In this series, leukocyte adhesion was reversed by a MAIE dose of 40 mg/rat, but not by a dose of 5 mg/rat. AlF at a dose of 0.01 mg per rat reversed neutrophil adhesion by approximately 25%. To further assess the effect of the higher concentration of MAIE (40 mg/rat), the number of neutrophils adhered at the beginning of the recording process and the number of new neutrophils adhered over 5 min at each time period were examined.

Figure 27a shows that less neutrophil adhesion occurred in the 10 min following MAIF administration, indicating that the anti-inflammatory factor had actually "dislodged" the adherent neutrophils. Furthermore, Figure 27b clearly shows that MAIF blocks new neutrophil adhesion to endothelial cells. The rate at which neutrophils rolled along the length of venules did not change between groups or with time, except that AlF may have increased the rolling speed of neutrophils (Figure 28). This effect was quite interesting given that red blood cell velocity remained unchanged (Figure 29). The results suggested that a simple increase in hydrodynamic forces cannot explain the increase in neutrophil rolling velocity. Neutrophil flux was also unaffected by MAIF, but was again reduced by AlF (Fig. 30).

The in vitro data show that the anti-inflammatory factor does not affect the activation of neutrophils per se. The superoxide scavenger superoxide dismutase completely blocks cytochrome C reduction by PMA- and fMLP-stimulated neutrophils, suggesting that this is a superoxide-mediated process. MAIF at extremely high concentrations only minimally affected cytochrome C reduction, suggesting that MAIF does not directly scavenge superoxide (Table 7). Protease release was not affected by MAIF (data not shown).

It was shown that the binding of monoclonal anti-CD18 antibodies could be reduced with MAIF or AlF (Table 8). This did not occur with the CD11b antibody. The binding of CD18 antibodies to IgG-coated Microbeads were also not affected by the MAIF or AIF preparations, suggesting that the anti-inflammatory factor did not affect the ability of the anti-CD18 monoclonal antibody to bind to substrate, but rather acted on the ligand CD18. The same pattern was observed with stimulated neutrophils (Table 9). It should be noted that binding to CD18 varied between days, as different cells were used each day. Therefore, a direct comparison of the results in Table 8 with those in Table 9 cannot be made.

Discussion:

The data in the above example suggest that the anti-inflammatory factor prevents neutrophil adhesion and emigration into venules in a dose-dependent manner. More importantly, the anti-inflammatory factor was able to reverse neutrophil adhesion to these vessels within a short period of time (10 min). The only other agents that can cause adherent neutrophils to release their adhesion to the endothelium with such efficiency are monoclonal antibodies directed against the CD11/CD18 glycoprotein complex on the neutrophil. MAIF appeared to have no effect on blood flow through the individual vessels or on systemic blood pressure, suggesting that hemodynamic factors such as shear stress could not explain the reversal of leukocyte adhesion. Although leukocyte rolling appears to be a prerequisite for leukocyte adhesion, MAIF did not affect leukocyte rolling velocity or flux. The latter result suggests that the anti-inflammatory factor did not affect the number of neutrophils rolling through the vessel and, therefore, that the reduction in adherent leukocytes was not a result of fewer leukocytes interacting with the endothelium. The fact that leukocyte rolling velocity as well as leukocyte flux remained unchanged suggests that adhesion molecules on neutrophils and endothelium that are responsible for leukocyte rolling (1L-selectin, P-selectin) were not affected by the anti-inflammatory factor in the MAIF preparation.

It has been reported that the leukocyte can regulate its own adhesion by releasing superoxide and also proteases. It was therefore conceivable that the lack of leukocyte adhesion in the presence of MAIF and AlF was due to the ability of these preparations to block the release of superoxide or proteases. This possibility is untenable in light of the fact that MAIF had little effect on superoxide or protease release and did not interact with released proteases or scavenge released superoxide. Moreover, MAIF did not appear to affect neutrophil viability as was observed with propidium iodide, making a direct cytotoxic effect of the anti-inflammatory factor on neutrophils unlikely.

A neutrophil must possess an intact CD11/CD18 glycoprotein complex to adhere and emigrate. Immunoneutralization of the adhesion complex completely reduces the ability of the neutrophil to adhere persistently to the endothelium and emigrate into the surrounding tissue. Since neutrophil adhesion and emigration is a rate-limiting step in tissue injury, in conjunction with a number of inflammatory conditions, an agent that impairs these processes would likely also block the inflammatory response. In the present study, both MAIF and AlF dramatically reversed neutrophil adhesion and blocked PAF-induced neutrophil emigration. Because of the similarity between the reversal of neutrophil adhesion induced by AlF, MAIF and monoclonal anti-CD18 antibodies, it seemed possible that the anti-inflammatory factor within AlF and MAIF exerted its effect by directly interacting with the CD18 glycoprotein complex. The in vitro data presented above support this view, in that both AlF and MAIF blocked the ability of an anti-CD18 antibody to bind to the CD18 glycoprotein complex. In contrast, Neither AlF nor MAIF inhibited the binding of CD11b to its corresponding monoclonal antibodies. Finally, AlF and MAIF preparations did not affect the ability of an anti-CD18 monoclonal antibody to bind to IgG-coated microbeads. Therefore, it can be concluded that the anti-inflammatory factor directly interacts with the CD18 complex and prevents CD18 from binding to various ligands including endothelial cell adhesion molecules.

Example 17 (comparative example) Effect of MAIF on circulating leukocytes

Some pharmacological agents can inhibit the migration of neutrophils. While some, such as cyclophosphamide, are cytoreductive and act by inhibiting hematopoiesis in the bone marrow, other agents, such as steroids and the non-steroidal anti-inflammatory drugs, have specific sites of action and do not induce leukocytosis. It was therefore important to determine the effect of the anti-inflammatory factor on the numbers and ratios of circulating white blood cells.

Two experiments were conducted. In the first, the MAIF preparation was administered intravenously at a dose of 40 mg/rat to a group of 6 animals and a control group was administered intravenously with saline. Blood samples were obtained at zero, 1, 4 and 24 hours after treatment. The results are summarized in Fig. 31.

MAIF administration resulted in an increase in the number of circulating neutrophils, maximum at 4 hours, and a corresponding reduction the number of peripheral blood lymphocytes. Another dose response study was conducted in which a group of rats was injected intravenously with saline, 5, 10 or 20 mg of the MAIF preparation. Blood was drawn from each rat 7 days before to provide baseline values and again 4 hours after injection of MAIF. The results are shown in Fig. 32. Included in the graph are the results of the sample taken 4 hours after administration of 40 mg of the MAIF preparation (see Fig. 31).

All doses of MAIF resulted in an increase in the number of circulating neutrophils and a decrease in the number of lymphocytes. While the effect on lymphocytes was linearly related to dose, the increase in the number of neutrophils was in the form of a curve, with the greatest effect observed in the animals given 10 mg.

These results support the concept that the anti-inflammatory factor modulates inflammation by influencing the adhesion of neutrophils to endothelial cells.

Data have also been obtained on the effects of three other cell-targeted anti-inflammatory/immunomodulatory agents on circulating leukocytes in the rat. The steroid drug methylprednisolone causes a change in the lymphocyte/neutrophil ratio analogous to that seen with MAIF. The temporal relationship between drug administration and effect is different. The antirejection/anti-inflammatory agent cyclosporin A also causes an increase in the number of circulating neutrophils, but depending on the dose, lymphocyte numbers are either increased or not affected. In In contrast, the cytotoxic drug cyclophosphamide reduces both circulating lymphocytes and neutrophils. The effects of the anti-inflammatory factor appear to modify the action of methylprednisolone.

Example 18 (comparative example) Effect of anti-inflammatory factor on lymphocyte function

The ability of the anti-inflammatory factor to induce a reversible reduction in the number of circulating lymphocytes (Example 17) prompted further investigation of the effect of the factor on lymphocyte function. Recipient versus host (GvH (graft vs host)) and host versus recipient (HvG (host vs graft)) analyses were used to determine the effect of the factor on T lymphocyte function.

In the HvG analysis, adult dark agouti rats (“DA”) were injected i.v. with 20 mg of the MAIF preparation 48, 24 and 3 hours before lymphocytes from their F1 hybrid progeny (DA · Hooded Oxford rats) were injected into their paws. Thus, the effect of the anti-inflammatory factor on the ability of intact host T lymphocytes (DA) to respond to the foreign histocompatibility antigens of the F1 lymphocytes was measured. The protocol produced a very significant reduction (30%) in the response, as evidenced by a reduction in popliteal lymph node weight (Fig. 33 A).

In the GvH reaction, parenteral (DA) lymphocytes were obtained from MAIF-treated parenteral rats (DA) and placed in the paws of their F1 (DA · Hooded Oxford) This assay measured the in vivo responsiveness of T lymphocytes taken from the host under evaluation, ie, MAIF-treated rats. The presence of MAIF had no effect on the GvH response (Fig. 33B).

During the previous experiments, an obvious increase in the number of spleen lymphocytes was noted in MAIF-treated animals. Further experiments showed a significant increase in both spleen weight and spleen cell number (Fig. 33C and 33D). The increase in spleen cell number was approximately equal to the previously reported decrease in circulating cell number.

Finally, the effect of the anti-inflammatory factor on the ability of isolated splenic lymphocytes to respond to the mitogenic concanavalin A was determined. Administration of the MAIF preparation showed an almost complete elimination of the mitogenic response of cultured lymphocytes to this lectin (Fig. 33E).

Example 19 (comparative example) Suppression of infection-induced inflammation by the anti-inflammatory factor

Experiments were conducted to determine whether changes in serum levels of acute phase reactants (APRs) could be used to quantify the anti-inflammatory activity of anti-inflammatory factor. The APRs are a group of proteins that are synthesized in response to an inflammatory stimulus. One of these, alpha-2-macroglobulin, is common to both humans and rats, and a methodology is available to measure this inflammatory component.

Two intravenous injections of a MAIF preparation (0 and 24 hours) did not reduce the peak response (48 hours) of alpha-2-macroglobulin. This result indicates that the factor does not affect the subsequent inflammatory response.

Example 20 (comparative example) In vitro and in vivo evaluation of milk-derived anti-inflammatory factor (Bovary Mammary Macrophage Assay, infection models in mice)

Incubation of bovine mammary macrophages with the hyperimmune milk fraction did not significantly improve the level of phagocytosis but increased the ability of macrophages to kill phagocytosed Staphylococcus aureus. Mice injected intraperitoneally with 10 mg of the MAIF preparation per kilogram demonstrated increased resistance to intraperitoneal challenge with lethal Staphylococcus aureus.

In an intramammary Staphylococcus aureus mastitis challenge model, MAIF injected into mice also showed markedly less mammary inflammation and involution and increased clearance of the infectious organism. Quantitative histological analysis of mammary tissue from MAIF-treated mice showed markedly more lumen, less interalveolar connective tissue, and less leukocyte infiltration than in mice. Mammary glands from treated mice also contained fewer colony forming units than control mice. The anti-inflammatory drug appears to exert its effect on the nonspecific defense system by modulating leukocyte function.

Example 21 (comparative example) Effect of the anti-inflammatory factor on the pathogenesis of an experimental infection

The most common inflammations in humans are microbial, and it is important to determine the effect of any agent that modulates the host defense against infection. Indeed, the tissue damage that accompanies many infectious diseases is caused by the host's response to infection rather than by the invading organism. While the ability to modulate the inflammatory response to infection could be a useful clinical tool, it must be recognized that inhibition of the host's response during infection can be detrimental. This is particularly true in the case of neutrophil inhibition. Studies using agents that reduce neutrophil involvement in the early stages of infection have shown that while inflammation and tissue damage can be initially suppressed, the increased bacterial load that occurs as a result of a reduced cellular response may eventually lead to an exacerbation of tissue damage. Thus, it is essential to determine the potential of milk anti-inflammatory factor to modulate inflammation to (1) determine whether the agent can reduce infection-induced tissue damage and (2) assess whether any observed suppression of the host response is accompanied by an increase in infection severity.

The effect of the anti-inflammatory factor on edema formation following intradermal injection of E. coli 075 was determined. Two groups of 8 animals were used. One group was untreated and served as a control while the individuals in the second group was injected intravenously with 40 mg of the MAIF preparation in 0.5 ml of saline. Immediately after administration of MAIF, 100 µl of an overnight culture of E. coli 075 was injected intradermally at two skin sites on the shaved back of the rat, followed by intradermal injection of 100 µl of saline at two additional sites. To allow an assessment of the volume of edema in the infected skin, 0.1 uCi of 125I-HSA was injected intravenously at the time of challenge. Six hours later, the animals were anesthetized, a blood sample obtained, the skin on the back removed, and the infected and saline-injected sites punctured. The volume of edema was calculated by relating tissue counts to plasma counts as described. To obtain the volume of edema accumulated as a result of the presence of E. coli, the edema/plasma volume of the saline-injected sites was subtracted. The results are shown in Fig. 34.

MAIF administration resulted in a 48% inhibition of edema formation. This experiment demonstrated that the anti-inflammatory factor could modulate the local inflammatory response to infection.

To study the relationships between anti-inflammatory factor administration, bacterial replication, fluid accumulation, and inflammatory cell infiltration, an alternative infection model was used. Polyurethane sponges prepared and implanted as previously described were infected with a quantified sample of E. coli 075 at the time of implantation. The sponges were removed at timed intervals, weighed to determine the volume of fluid exudate, and then compressed in a medium to remove the bacteria and cells from the sponge. Bacteria and cell numbers were estimated using methods known to those of ordinary skill in the art. The following experiment was performed using this model. Ninety animals were divided into two groups of 45. One of these groups was untreated and served as a control. The second group was injected intravenously with 40 mg of the MAIF preparation. The sponges were then implanted subcutaneously and each sponge was inoculated with 10⁵ E. coli 075 at the time of implantation. Groups of 6-8 animals were sacrificed at intervals thereafter and the bacteriological status and size of the inflammatory infiltrate in the sponges were determined. The results are illustrated in Figs. 35-37.

The rate of bacterial replication was much greater in MAIF-treated animals than in controls, and there was a 10, 1000, and 10,000-fold difference in bacterial counts at 4, 8, and 16 hours, respectively. Thereafter, bacterial counts decreased, although a large difference was still present at 96 hours (Fig. 35).

The early response to infection is the critical determinant in the outcome of an infectious event. In this experiment, the cellular infiltrate at 2, 4, and 8 h in those animals given MAIF was 27%, 35%, and 46% of the control infiltrate, respectively (Fig. 36B). The cells that accumulated in the first 24 h after challenge are >90% neutrophilic leukocytes, and suppression of this cellular component during this phase may explain the rapid increase in bacterial numbers. Fluid accumulation at 2 h was not affected by MAIF administration, but was markedly lower at 4, 8, and 16 h after challenge. This is consistent with the previous finding that the antiinflammatory factor induces the primary, noncellular phase. of edema formation in the carrageenan paw model. In previous studies using the immunomodulators cyclosporin A and methylprednisolone, a similar relationship was found between suppression of the acute cellular inflammatory infiltrate and promotion of bacterial replication. However, in these experiments, the increased bacterial load promoted a host response between 24 and 48 hours after challenge in which there was a massive influx of neutrophils. When tissue was involved, the enhanced inflammatory response resulted in a marked exacerbation of tissue damage and scarring. Interestingly, although administration of MAIF suppressed the early inflammatory response and was associated with a 10,000-fold increase in bacterial numbers, there was no massive influx of neutrophils 24-48 hours after challenge.

Example 22 (comparative example) Effect of anti-inflammatory factor on experimental pyelonephritis

An agent that can suppress inflammation in infection without the sequela of increased tissue damage would have considerable potential. A clinically relevant model of infectious disease could provide an experimental basis for establishing such potential.

Pyelonephritis is an infectious disease that shows local inflammation, tissue destruction and scarring as the main histological features. A well-characterized model of the disease is available that reproduces the central pathological features of the disease in humans. Pyelonephritis is rat by direct inoculation of the surgically exposed kidney with a predetermined number of E. coli 075; following challenge, bacterial numbers increase rapidly, reaching a peak 3 to 4 days later. In normal animals, the level of infection falls over the next 5 or 6 days, reaching a plateau at approximately 10 days after challenge. By 21 days, the lesions have resolved to represent focal areas of depressed scar tissue. To assess the effect of anti-inflammatory factor on this pattern of infection, pyelonephritis was induced in both kidneys of twenty-six animals. One-half of these animals were treated intravenously with the MAIF preparation at a dose of 40 mg/rat at the time of challenge and again 48 hours later. Seven animals from each group were sacrificed 4 days after induction of pyelonephritis and the two remaining groups of six animals at 21 days. The kidneys were aseptically removed and weighed to determine the relative volume of fluid exudate. The extent of surface lesion size was estimated by direct observation and the kidney was homogenized to allow determination of bacterial numbers. The results are shown in Fig. 38.

Four days after challenge, the inflammatory response was suppressed by MAIF administration, as evidenced by inhibition of fluid accumulation and the size of lesions on the surface of the kidney. As previously observed in studies involving infected, subcutaneously implanted sponges, early suppression of inflammation resulted in a logarithmic increase in the number of bacteria in MAIF-treated animals. At 21 days, there was no difference in disease pathology as measured by kidney weight, bacterial counts, or lesion sites of the renal surface. Thus, while suppression of the early inflammatory response by MAIF did not reduce tissue destruction in the chronic (21 day) phase of pyelonephritis, it did not promote the development of pathological lesions, as did other anti-inflammatory and immunomodulatory agents.

Example 23 (comparative example) Summary of experimental data

A method has been developed that allows for the continuous monitoring of edema accumulation in carrageenan-injected paws.

The early phase, nonphagocytic phase of the inflammatory response was not affected by any anti-inflammatory factor, while the later, cellular-driven phase of the response was significantly inhibited. Further experiments in which MAIF was administered at intervals before or after carrageenan injection provided additional evidence that MAIF exerts its anti-inflammatory effect by modulating the secondary, neutrophil-mediated inflammatory response.

It was shown that the anti-inflammatory factor had a half-life of 1-2 hours after i.v. injection and that development of inflammation could be suppressed when the factor was administered 30 minutes after irritation. This result is relevant for the potential therapeutic use of the anti-inflammatory factor.

The neutrophil is the main cell involved in the acute inflammatory response. During the Arthus reaction, a reduction in neutrophil accumulation > 80% was observed following MAIE administration, which in turn is associated with a very significant inhibition of the secondary features of the inflammatory response, namely edema and hemorrhage. This result further makes neutrophil a target in MAIF-induced suppression of inflammation.

One of the key steps in the development of inflammation is the migration of neutrophils from the vasculature to the tissues. Intravenous administration of the anti-inflammatory factor resulted in a pronounced and dose-dependent inhibition of neutrophil migration. When the effect of the anti-inflammatory factor on peripheral blood leukocytes was investigated, a pronounced increase in the number of circulating leukocytes was observed, accompanied by a corresponding decrease in the number of lymphocytes. This effect was also dose-dependent, but was not linear in the case of the increase in the number of neutrophils.

Administration of anti-inflammatory factor from milk was shown to block the adhesion of neutrophils to the endothelium and promote the dissociation of neutrophils that were adherent at the time of administration. This effect is probably the result of the ability of the anti-inflammatory factor to block the interaction between cell surface CD18 antigens and other molecules. The inhibition of CD18 binding by the factor appears to be specific, in that the factor inhibits the binding of monoclonal anti-CD18 antibodies to cells. but did not equally prevent the binding of the monoclonal anti CD11b antibodies.

Blocking the intermolecular interactions involving the CD18 cell surface antigen may also explain the observation that the factor could inhibit the ability of host lymphocytes to respond to foreign histocompatibility antigens. In other experiments, the anti-inflammatory factor was shown to block the concanavalin-induced mitogenic response in lymphocytes.

Finally, the factor markedly suppresses the early cellular response to infection, an effect that resulted in a logarithmic increase in bacterial numbers in a model of subcutaneous infection. This exacerbation of infection did not result in a relapse of the inflammatory response as seen with other agents that suppress acute inflammation in infection. A second experiment using a clinically relevant infection model, pyelonephritis, also demonstrated a suppressive effect on inflammation that was associated with an increase in bacterial numbers. Again, no relapse effect was observed and there was no difference in the degree of tissue damage that occurred in MAIF-treated and control groups.

The following conclusions can be drawn from this series of experiments:

1. An i.v. administered anti-inflammatory factor suppresses the secondary, neutrophil-mediated phase of the carrageenan-induced inflammatory response.

2. When evaluated in the carrageenan paw assay, the anti-inflammatory factor has a biological half-life of 1-2 hours and is effective even when administered after inflammation is induced. Subsequent experiments show that the effective half-life is dependent on both the dose used and the inflammatory stimulus.

3. The anti-inflammatory factor inhibits the emigration of neutrophil leukocytes in vivo.

4. Administration of the anti-inflammatory factor results in an increase in the number of circulating leukocytes and a corresponding decrease in lymphocyte numbers.

5. The anti-inflammatory factor suppresses host defense against infection, probably via an effect on the emigration of neutrophils.

6. The anti-inflammatory factor blocks the interactions between cell surface CD18 antigens and other molecules.

7. The anti-inflammatory factor blocks the adhesion of neutrophil leukocytes to the endothelium.

8. The anti-inflammatory factor promotes the dissociation of adherent neutrophils from the endothelium.

9. The anti-inflammatory factor blocks the ability of host lymphocytes to respond to foreign histocompatibility antigens.

10. The anti-inflammatory factor blocks the mitogenic response of lymphocytes.

The experimental data obtained in these studies clearly demonstrate that the milk anti-inflammatory factor has a pronounced effect on both neutrophilic leukocytes and lymphocytes. The observed effects may be the result of a direct effect of the anti-inflammatory factor on cells per se or the result of the suppression (or stimulation) of some other cellular or soluble mediators which indirectly alter the biological activities of cells. It is also widely accepted that most pharmacological agents have multiple effects, and it is possible that the anti-inflammatory factor may turn out to affect a number of other, as yet unknown, biological processes.

Having now generally described this invention, it will be readily apparent to one of ordinary skill in the art that many changes and modifications may be made therein without affecting the spirit or scope thereof.

Claims (16)

1. A composition comprising an anti-inflammatory factor, wherein the anti-inflammatory factor is at least about 55,000-fold more purified than the factor in a 10,000 dalton skim milk filtrate tested in the mouse neutrophil migration inhibition assay, the composition being prepared by a process comprising: (a) removing fat from the milk of a milk-producing animal to produce a skimmed-milk solution; b) filtering the skim milk solution from step a) through a filter to produce a skim milk filtrate having a molecular weight ≤ 10,000, wherein the filter retains molecules having molecular weights greater than about 10,000 Daltons; c) fractionating the skimmed milk filtrate produced in step b by ion exchange chromatography to produce ion exchange fractions enriched in the factor; d) purifying the factor from the enriched ion exchange fractions by gel filtration chromatography to produce gel filtration fractions further enriched in the factor; e) purifying the factor from the enriched gel filtration fractions by affinity chromatography to produce a composition containing the anti-inflammatory factor which 55,000-fold more purified than the factor in the skim milk filtrate from step b, tested in the mouse neutrophil migration inhibition assay. 2. The composition of claim 1, wherein the affinity chromatography is carried out on an affinity medium which has an affinity for coplanar adjacent cis-hydroxyl groups. 3. A composition for detaching neutrophils attached to endothelial cells in a mammal, which comprises the composition according to claim 1 or 2 as an active ingredient. 4. The composition of claim 3, wherein the neutrophils have attached to the endothelial cells in response to a platelet-activating factor. 5. A composition for detaching neutrophils according to claim 3 which have attached to endothelial cells in a mammal, the composition being prepared by a process comprising: (i) removing fat from milk of a milk-producing animal to produce skimmed milk; (ii) pasteurizing the skimmed milk; (iii) removing casein from the pasteurised skim milk to produce whey; (iv) removing macromolecules having a molecular weight greater than about 10,000 Daltons from the whey to produce a composition free of macromolecules having a molecular weight greater than about 10,000 Daltons; (v) reducing the ionic strength of the composition of step (iv) to produce an aggregate having anti-inflammatory activity, wherein the aggregate has a molecular weight of greater than about 5000 Daltons; (vi) removing macromolecules having a molecular weight of less than about 5,000 daltons from the composition of step (v) to produce a composition free of macromolecules having a molecular weight of less than about 5,000 daltons; (vii) collecting the composition from step (vi). 6. A composition for preventing the interaction between CD18 cell surface atigens present in a mammal and other molecules, comprising the composition according to claim 1 or 2 as an active ingredient. 7. A composition for preventing the interaction between CD18 cell surface atigens found in a mammal and other molecules according to claim 6, wherein the composition is prepared by a process comprising: (i) removing fat from milk of a milk-producing animal to produce skimmed milk; (ii) pasteurizing the skimmed milk; (iii) removing casein from the pasteurised skim milk to produce whey; (iv) removing macromolecules having a molecular weight greater than about 10,000 daltons from the whey to produce a composition free of macromolecules having a molecular weight greater than about 10,000 daltons; (v) reducing the ionic strength of the composition of step (iv) to produce an aggregate having anti-inflammatory activity, wherein the aggregate has a molecular weight of greater than about 5000 Daltons; (vi) removing macromolecules having a molecular weight of less than about 5,000 daltons from the composition of step (v) to produce a composition free of macromolecules having a molecular weight of less than about 5,000 daltons; (vii) collecting the composition from step (vi). 8. A composition for preventing the emigration of cells of the venous system, which comprises the composition according to claim 1 or 2 as an active ingredient. 9. The composition of claim 8, wherein the cells are leukocytes. 10. The composition of claim 8, wherein the cells are neutrophils. 11. Composition for preventing the emigration of cells of the venous system according to claim 8, wherein the composition is prepared by a process comprising: (i) removing fat from milk of a milk-producing animal to produce skimmed milk; (ii) pasteurizing the skimmed milk; (iii) removing casein from the pasteurised skim milk to produce whey; (iv) removing macromolecules having a molecular weight greater than about 10,000 daltons from the whey to produce a composition free of macromolecules having a molecular weight greater than about 10,000 daltons; (v) reducing the ionic strength of the composition of step (iv) to produce an aggregate having anti-inflammatory activity, wherein the aggregate has a molecular weight of greater than about 5000 Daltons; (vi) removing macromolecules having a molecular weight of less than about 5,000 daltons from the composition of step (v) to produce a composition free of macromolecules having a molecular weight of less than about 5,000 daltons; (vii) collecting the composition from step (vi). 12. A composition for suppressing the mitogenic response of lymphocytes in a host mammal to foreign antigens, which comprises the composition according to claim 1 or 2 as an active ingredient. 13. The composition of claim 12, wherein the antigens are located on the surface of cells other than the cells of the host. 14. The composition of claim 13, wherein the cells are leukocytes. 15. The composition of claim 13, wherein the cells are lymphocytes. 16. A composition for suppressing the mitogenic response of lymphocytes in a host mammal to foreign antigens according to claim 12, wherein the composition is prepared by a process comprising: (i) removing fat from milk of a milk-producing animal to produce skimmed milk; (ii) pasteurizing the skimmed milk; (iii) removing casein from the pasteurised skim milk to produce whey; (iv) removing macromolecules having a molecular weight greater than about 10,000 daltons from the whey to produce a composition free of macromolecules having a molecular weight greater than about 10,000 daltons; (v) reducing the tonicity of the composition of step (iv) to produce an aggregate having anti-inflammatory activity, wherein the aggregate has a molecular weight of greater than about 5000 Daltons; (vi) removing macromolecules having a molecular weight of less than about 5,000 daltons from the composition of step (v) to produce a composition free of macromolecules having a molecular weight of less than about 5,000 daltons; (vii) collecting the composition from step (vi).